Bispecific anti-vegf/anti-ang-2 antibodies and their use in the treatment of ocular vascular diseases

ABSTRACT

The present invention relates to bispecific antibody against human vascular endothelial growth factor (VEGF/VEGF-A) and against human angiopoietin-2 (ANG-2) of human IgG1 or IgG4 subclass with mutations I253A, H310A, and H435A, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/603,259, filed May 23, 2017 which is a continuation of U.S.application Ser. No. 13/940,091, filed Jul. 11, 2013, now U.S. Pat. No.9,695,233, which claims the benefit of priority under 35 USC 119(a) toEuropean patent application number 12176299.1, filed 13 Jul. 2012, whichare incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 18, 2020, isnamed Sequence_listing.txt and is 120,540 bytes in size.

FIELD OF THE INVENTION

The present invention relates a method for the reduction of theviscosity of an antibody (including a bispecific antibody) of human IgG1or human IgG4 subclass, to bispecific antibodies against human vascularendothelial growth factor (VEGF/VEGF-A) and against human angiopoietin-2(ANG-2), methods for their production, pharmaceutical compositionscontaining said antibodies, and uses thereof.

BACKGROUND OF THE INVENTION

Angiogenesis is implicated in the pathogenesis of a variety of disorderswhich include solid tumors, intraocular neovascular syndromes such asproliferative retinopathies or age-related macular degeneration (AMD),rheumatoid arthritis, and psoriasis (Folkman, J., et al., J. Biol. Chem.267 (1992) 10931-10934; Klagsbrun, M., et al., Annu. Rev. Physiol. 53(1991) 217-239; and Garner, A., Vascular diseases, in: Pathobiology ofocular disease, A dynamic approach, Garner, A., and Klintworth, G. K.(eds.), 2nd edition, Marcel Dekker, New York (1994), pp. 1625-1710).

Ranibizumab (trade name Lucentis®) is a monoclonal antibody fragmentderived from the same parent murine antibody as bevacizumab (Avastin®).However, it has been affinity matured to provide stronger binding toVEGF-A (WO 98/45331). It is known that VEGF-A blocking may be related tosome systemic toxicities, therefore ranibizumab is missing an Fc part toreduce the serum half live and consequently systemic toxicities. It isan anti-angiogenic agent that has been approved to treat the “wet” typeof age-related macular degeneration (ARMD), a common form of age-relatedvision loss.

Corneal angiogenesis assays have shown that both ANG-1 and ANG-2 hadsimilar effects, acting synergistically with VEGF to promote growth ofnew blood vessels. Asahara, T., et al., Circ. Res. 83 (1998) 233-40. Thepossibility that there was a dose-dependent endothelial response wasraised by the observation that in vitro at high concentration, ANG-2 canalso be pro-angiogenic (Kim, I., et al., Oncogene 19 (2000) 4549-52). Athigh concentration, ANG-2 acts as an apoptosis survival factor forendothelial cells during serum deprivation apoptosis through activationof Tie2 via PI-3 Kinase and Akt pathway (Kim, I., et al., Oncogene 19(2000) 4549-52).

WO 2010/040508 A9 and WO 2011/117329 relate to bispecificanti-VEGF/anti-ANG-2 antibodies. WO 2008/132568 relates to fusionproteins binding to growth factors. WO 2009/136352 relates toanti-angiogenic compounds. WO 2009/080253 and WO 2011/117330 relates tobispecific bivalent antibody formats. WO 2010/069532 relates to Ang2antibodies.

Ocular vascular diseases such as age related macular degeneration (ARMD)and diabetic retinopathy (DR) are due to abnormal choroidal or retinalneovascularization respectively. They are the leading causes of visualloss in industrialized nations. Since the retina consists ofwell-defined layers of neuronal, glial, and vascular elements,relatively small disturbances such as those seen in vascularproliferation or edema can lead to significant loss of visual function.Inherited retinal degenerations, such as Retinitis Pigmentosa (RP), arealso associated with vascular abnormalities, such as arteriolarnarrowing and vascular atrophy. They affect as many as 1 in 3500individuals and are characterized by progressive night blindness, visualfield loss, optic nerve atrophy, arteriolar attenuation, and centralloss of vision often progressing to complete blindness.

Ischemic retinopathies are characterized by loss or dysfunction of theretinal vasculature which results in a reduction of blood flow andhypoxia. The retina responds to hypoxia by generating signals to grownew blood vessels, but these new vessels are usually fragile anddisorganized. It is the growth of these abnormal new vessels thatcreates most of the threat to vision since they can leak, hemorrhage orlead to scarring that may end in retinal detachment. Current treatmentsfor ischemic retinopathies seek to halt the growth of the pathologicalvessels but do not address the underlying ischemia that drives theirgrowth. Furthermore, standard treatment for diabetic retinopathy, anischemic retinopathy that affects millions, involves destruction of aportion of the retina with a laser in an attempt to stop new vesselgrowth and preserve central vision. Strategies have been employed toblock the function of vascular endothelial growth factor (VEGF), a majorpromoter of vessel growth. In the short term, anti-VEGF therapy canimprove vision, but it does not address the underlying ischemia and infact may exacerbate this condition as it inhibits all vessel growth,including beneficial collaterals. There is also the serious concern ofsystemic exposure of these drugs in elderly and/or diabetic patientswhere new vessel growth may be required in ischemic brains, hearts orlimbs.

Typically for ocular diseases via intravitreal application smallerantibody fragments like Fab or Fab(2) are often used as they have a lowserum half-life and the risk of systemic toxicities is lower. Howeverthis smaller fragments typically have also lower intravitreal half-lifes(e.g. due to the faster diffusion into serum) and have to be dosedtypically more often.

Kim et al, Molecular Vision, 15 (2009) 2803-2812 relates to full lengthantibodies administered intravitreally in the eye, wherein an IgG withFcRn binding was eliminated into the blood in wild-type mice, whereas anIgY with no FcRn binding was not eliminated into the blood system.Furthermore the IgG with FcRn binding was not eliminated into the bloodsystem in FcRn knockdown-mice.

There is a need in the art for better means for treating and preventingvarious ocular vascular diseases such as ischemic retinopathies.

SUMMARY OF THE INVENTION

One aspect of the invention is method for the reduction of the viscosityof an antibody wherein the antibody comprises a constant heavy chainregion of human IgG1 or human IgG4 subclass (derived from human originand) wherein the method comprises the modification of the antibodyconstant heavy chain region of human IgG1 or human IgG4 subclass withthe mutations I253A, H310A, and H435A (numbering according to EU Indexof Kabat).

In one embodiment of the invention said method is characterized in thatthe antibody is a bispecific antibody comprising a first antigen-bindingsite that specifically binds to human VEGF and a second antigen-bindingsite that specifically binds to human ANG-2, wherein

-   -   i) said first antigen-binding site specifically binding to VEGF        comprises in the heavy chain variable domain a CDR3H region of        SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region        of SEQ ID NO:3, and in the light chain variable domain a CDR3L        region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a        CDR1L region of SEQ ID NO:6; and    -   ii) said second antigen-binding site specifically binding to        ANG-2 comprises in the heavy chain variable domain a CDR3H        region of SEQ ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a        CDR1H region of SEQ ID NO: 11, and in the light chain variable        domain a CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID        NO: 13, and a CDR1L region of SEQ ID NO: 14, and wherein    -   iii) the bispecific antibody comprises a constant heavy chain        region of human IgG1 or human IgG4 subclass (derived from human        origin and) comprising the mutations I253A, H310A, and H435A        (numbering according to EU Index of Kabat).

In one embodiment of the invention such method is characterized in thatsaid bispecific antibody described above comprises a constant heavychain region of human IgG1 subclass (derived from human origin and)comprising the mutations I253A, H310A, and H435A (numbering according toEU Index of Kabat) and further comprising the mutations L234A, L235A andP329G (numbering according to EU Index of Kabat).

One embodiment of the invention is an antibody obtained by such method.

One embodiment of the invention is the use of the mutations I253A,H310A, and H435A (numbering according to EU Index of Kabat) for thereduction of the viscosity of an antibody wherein the antibody comprisesa constant heavy chain region of human IgG1 or human IgG4subclass(derived from human origin).

In one embodiment of the invention said use is characterized in that theantibody is a bispecific antibody comprising a first antigen-bindingsite that specifically binds to human VEGF and a second antigen-bindingsite that specifically binds to human ANG-2,

wherein

-   -   i) said first antigen-binding site specifically binding to VEGF        comprises in the heavy chain variable domain a CDR3H region of        SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region        of SEQ ID NO:3, and in the light chain variable domain a CDR3L        region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a        CDR1L region of SEQ ID NO:6; and    -   ii) said second antigen-binding site specifically binding to        ANG-2 comprises in the heavy chain variable domain a CDR3H        region of SEQ ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a        CDR1H region of SEQ ID NO: 11, and in the light chain variable        domain a CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID        NO: 13, and a CDR1L region of SEQ ID NO: 14, and wherein    -   iii) the bispecific antibody comprises a constant heavy chain        region of human IgG1 or human IgG4 subclass (derived from human        origin and) comprising the mutations I253A, H310A, and H435A        (numbering according to EU Index of Kabat).

In one embodiment of the invention said specific use is characterized inthat the bispecific antibody comprises a constant heavy chain region ofhuman IgG1 subclass (derived from human origin and) comprising themutations I253A, H310A, and H435A (numbering according to EU Index ofKabat) and further comprising the mutations L234A, L235A and P329G(numbering according to EU Index of Kabat).

The invention is further directed to a bispecific, bivalent antibodycomprising a first antigen-binding site that specifically binds to humanVEGF and a second antigen-binding site that specifically binds to humanANG-2, wherein

-   -   i) said first antigen-binding site specifically binding to VEGF        comprises in the heavy chain variable domain a CDR3H region of        SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region        of SEQ ID NO:3, and in the light chain variable domain a CDR3L        region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a        CDR1L region of SEQ ID NO:6; and    -   ii) said second antigen-binding site specifically binding to        ANG-2 comprises in the heavy chain variable domain a CDR3H        region of SEQ ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a        CDR1H region of SEQ ID NO: 11, and in the light chain variable        domain a CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID        NO: 13, and a CDR1L region of SEQ ID NO: 14,

and wherein

-   -   iii) the bispecific antibody comprises a constant heavy chain        region of human IgG1 or human IgG4 subclass (derived from human        origin and) comprising the mutations I253A, H310A, and H435A        (numbering according to EU Index of Kabat).

In one embodiment said bispecific antibody is characterized in that

-   -   i) said first antigen-binding site specifically binding to VEGF        comprises as heavy chain variable domain VH an amino acid        sequence of SEQ ID NO: 7, and as light chain variable domain VL        an amino acid sequence of SEQ ID NO: 8, and    -   ii) said second antigen-binding site specifically binding to        ANG-2 comprises as heavy chain variable domain VH an amino acid        sequence of SEQ ID NO: 15, and as light chain variable domain VL        an amino acid sequence of SEQ ID NO: 16.

In one embodiment said bispecific antibody is characterized in that theconstant heavy chain region under iii) is of human IgG1 subclass. In oneembodiment said bispecific antibody of IgG1 subclass is characterized inthat the constant heavy chain region of IgG1 subclass further comprisesthe mutations L234A, L235A and P329G (numbering according to EU Index ofKabat).

In one embodiment said bispecific antibody is characterized in that theconstant heavy chain region under iii) is of human IgG4 subclass. In oneembodiment said bispecific antibody of IgG4 subclass is characterized inthat the constant heavy chain region of IgG4 subclass further comprisesthe mutations S228P and L235E (numbering according to EU Index ofKabat). In one embodiment said bispecific antibody of IgG4 subclass ischaracterized in that the constant heavy chain region of IgG4 subclassfurther comprises the mutations 228P, L235E and P329G (numberingaccording to EU Index of Kabat).

Still further aspects of the invention are a pharmaceutical compositioncomprising said bispecific antibody, said pharmaceutical composition foruse in the treatment of ocular vascular diseases, the use of saidbispecific antibody for the manufacture of a medicament for thetreatment of ocular vascular diseases, a method of treatment of patientsuffering from ocular vascular diseases by administering said bispecificantibody to a patient in the need of such treatment. In one embodimentthe bispecific antibody or the pharmaceutical composition comprisingsaid bispecific antibody is administered via intravitreal application.

A further aspect of the invention is a nucleic acid molecule encoding aheavy and/or light chain of a bispecific antibody according to theinvention.

The invention further provides expression vectors containing saidnucleic acid according to the invention capable of expressing saidnucleic acid in a prokaryotic or eukaryotic host cell, and host cellscontaining such vectors for the recombinant production of a bispecificantibody according to the invention.

The invention further comprises a prokaryotic or eukaryotic host cellcomprising a vector according to the invention.

The invention further comprises a method for the production of abispecific antibody according to the invention, characterized byexpressing a nucleic acid according to the invention in a prokaryotic oreukaryotic host cell and recovering said bispecific antibody from saidcell or the cell culture supernatant. One embodiment is a method for thepreparation of a bispecific antibody according to the inventioncomprising the steps of

a) transforming a host cell with vectors comprising nucleic acidmolecules encoding said antibody;

b) culturing the host cell under conditions that allow synthesis of saidantibody molecule; and

c) recovering said antibody molecule from said culture.

The invention further comprises the antibody obtained by such method forthe production of a bispecific antibody.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 21, of SEQ ID NO: 22, of SEQ ID NO: 23, and of SEQ ID NO: 24.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 25, of SEQ ID NO: 26, of SEQ ID NO: 27, and of SEQ ID NO: 28.

The antibodies according to the invention have highly valuableproperties due to their specific modifications in the Fc part/constantregion causing a benefit for a patient suffering from ocular vasculardiseases. They show high stability in the intravitreal environment andslow diffusion from the eye (compared to smaller antibody fragmentswithout a constant heavy chain region), where the actual disease islocated and treated (so treatment schedule can potentially be improvedcompared to non-IgG like antibodies like e.g. Fab and (Fab)2 fragments).Surprisingly compared to unmodified IgG antibodies the half-life in theeye after intravitreal application of the antibodies with the mutationsI253A, H310A, and H435A in the constant region (with no more FcRnbinding) was similar (only slightly reduced) (Tables 17a and 18a andFIGS. 7D and 7E), whereas the diffusion from the eye into the bloodserum was similar (Table 15 and FIG. 7B). This highly valuable as it isdesired for the treatment of ocular vascular diseases related to ANG2and/or VEGF it to eliminate VEGF and Ang2 from the eye (e. via thetransportation into the blood serum as anti-ANG2/ANG2 antibody complexor anti-VEGF/VEGF antibody complex). The antibodies according to theinvention are cleared on the other hand quite rapidly from serum whencompared to unmodified IgG antibodies (which is highly desired to reducepotential side effects arising from systemic exposure).

Surprisingly they also show lower viscosity (see FIG. 2) (compared toversions without the mutations I253A, H310A, and H435A in the constantregion) and are therefore especially useful for intravitreal applicationthrough thin needles during the treatment of eye diseases (for suchapplication typically thin needles are used and high viscosity makes anappropriate application rather difficult). The lower viscosity alsoallows higher concentration formulations.

Also surprisingly the antibodies according to the invention show a loweraggregation tendency (FIG. 4) during storage (compared to versionswithout the mutations I253A, H310A, and H435A in the Fc part) which iscritical for intravitreal application in the eye (as an aggregation inthe eye can lead to complications during such treatment). The bispecificantibodies according to the invention show good efficacy in inhibitionof vascular diseases.

In certain embodiments, the bispecific antibodies according to theinvention due to their specific modifications in the constant region(e.g. P329G LALA) show valuable properties like no binding Fcgammareceptors which reduces the risk of side effects like thrombosis and/orunwanted cell death (due to e.g. ADCC).

DESCRIPTION OF THE FIGURES

FIG. 1 Scheme of concept and advantages of <VEGF-ANG-2> IgG1 or IgG4antibodies with AAA mutations (mutations I253A, H310A, and H435A-numbering according to EU Index of Kabat).

FIG. 2 Small-scale DLS-based viscosity measurement Extrapolatedviscosity at 150 mg/mL in 200 mM Arginine/Succinate, pH 5.5 (comparisonof <VEGF-ANG-2> antibodies according to the invention VEGFang2-0016(with AAA mutations) with a reference VEGFang2-0015 (without such AAAmutations).

FIG. 3 DLS Aggregation depending on temperature (including DLSaggregation onset temperature) in 20 mM His, 140 mM NaCl, pH 6.0 5(comparison of <VEGF-ANG-2> antibodies according to the inventionVEGFang2-0016 (with AAA mutations) with a reference VEGFang2-0015(without such AAA mutations).

FIG. 4 7 day storage at 40° C. at 100 mg/ml (Decrease of Main and HighMolecular Weight/HMW) increase) (comparison of <VEGF-ANG-2> antibodiesaccording to the invention VEGFang2-0016 (with AAA mutations) whichshowed a lower aggregation with a reference VEGFang2-0015 (without suchAAA mutations)).

FIG. 5A FcRn steady state affinity of VEGFang2-0015 (without AAAmutations): overlay of Biacore sensogramms at different concentrationsshows a concentration dependent binding of VEGFang2-0015 (without AAAmutations) to FcRn.

FIG. 5B FcRn steady state affinity of A: VEGFang2-0015 (without AAAmutations): the concentration dependent binding response curve ofVEGFang2-0015 (without AAA mutations) shows binding to FcRn.

FIG. 5C FcRn steady state affinity of VEGFang2-0016 (with AAAmutations): overlay of Biacore sensogramms at different concentrationsshows no binding to FcRn at all concentrations.

FIG. 5D FcRn steady state affinity of VEGFang2-0016 (with AAAmutations): the concentration dependent binding response curve ofVEGFang2-0016 (with AAA mutations) shows no binding to FcRn.

FIG. 5E FcRn steady state affinity of VEGFang2-0016 (with AAAmutations): the concentration dependent binding response curve ofVEGFang2-0016 (with AAA mutations) shows no binding to FcRn (Responserange from -0.6 to 0.2 RU/concentration scale ranges from 0 to 0.35 M).

FIG. 6 FcgammaRIIIa interaction of VEGFang2-0015 without AAA mutationsand VEGFang2-0016 with AAA mutations measurement (both are IgG1 subclasswith P329G LALA mutations; as controls an Anti-Dig of IgG1 subclass anda IgG4 based antibody was used).

FIG. 7A Schematic Pk-ELISA Assay Principle for determination ofconcentrations of <VEGF/Ang2> bispecific antibodies in serum and wholeeye lysates.

FIG. 7B Serum concentration after intravenous application: Comparison ofcompounds -VEGFang2-0015 without AAA mutations and VEGFang2-0016 withAAA mutations.

FIG. 7C Serum concentration after intravitreal application: Comparisonof compounds -VEGFang2-0015 without AAA mutations and VEGFang2-0016 withAAA mutations.

FIG. 7D Eye lysates concentration of VEGFang2-0016 (with AAA mutation)in right and left eye (after intravitreal application only into theright eye in comparison to intravenous application): Significantconcentrations could be detected only in the right eye afterintravitreal application. After intravenous application noconcentrations in eye lysates could be detected due to the low serumhalf-life of VEGFang2-0016 (with AAA mutation).

FIG. 7E Eye lysates concentration of VEGFang2-0015 (without AAAmutation) in right and left eye (after intravitreal application onlyinto the right eye in comparison to intravenous application): In theright eye (and to some extent in the left eye) after intravitrealapplication concentrations of VEGFang2-0015 could be detected. Thisindicates the diffusion from the right eye into serum and from thereinto the left eye, which can be explained by the long half-life ofVEGFang2-0015 (without AAA mutation). After intravenous application alsosignificant concentrations in eye lysates of both eyes could be detecteddue to diffusion into the eyes of the serum-stable VEGFang2-0015(without AAA mutation).

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the invention the bispecific antibody according tothe invention is bivalent.

In one aspect of the invention such bispecific, bivalent antibodyaccording to the invention is characterized in comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to VEGF;    -   b) the modified heavy chain and modified light chain of a second        full length antibody that specifically binds to ANG-2, wherein        the constant domains CL and CH1 are replaced by each other.

This bispecific, bivalent antibody format for the bispecific antibodyspecifically binding to human vascular endothelial growth factor (VEGF)and human angiopoietin-2 (ANG-2) is described in WO 2009/080253(including Knobs-into-Holes modified CH3 domains). The antibodies basedon this bispecific, bivalent antibody format are named CrossMabs.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 25, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 27,        and    -   b) as modified heavy chain of the second full length antibody        the amino acid sequence of SEQ ID NO: 26, and as modified light        chain of the second full length antibody the amino acid sequence        of SEQ ID NO: 28.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 21, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 23,        and    -   b) as modified heavy chain of the second full length antibody        the amino acid sequence of SEQ ID NO: 22, and as modified light        chain of the second full length antibody the amino acid sequence        of SEQ ID NO: 24.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 29, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 31,        and    -   b) as modified heavy chain of the second full length antibody        the amino acid sequence of SEQ ID NO: 30, and as modified light        chain of the second full length antibody the amino acid sequence        of SEQ ID NO: 32.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 25, of SEQ ID NO: 26, of SEQ ID NO: 27, and of SEQ ID NO: 28.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 21, of SEQ ID NO: 22, of SEQ ID NO: 23, and of SEQ ID NO: 24.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 29, of SEQ ID NO: 30, of SEQ ID NO: 31, and of SEQ ID NO: 32.

In another aspect of the invention the bispecific antibody according tothe invention is characterized in comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to VEGF;    -   b) the heavy chain and the light chain of a second full length        antibody that specifically binds to ANG-2, wherein the        N-terminus of the heavy chain is connected to the C-terminus of        the light chain via a peptide linker.

This bispecific, bivalent antibody format for this bispecific antibodyspecifically binding to human vascular endothelial growth factor (VEGF)and human angiopoietin-2 (ANG-2) is described in WO 2011/117330including Knobs-into-Holes modified CH3 domains. The antibodies based onthis bispecific, bivalent antibody format are named OAscFabs.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 33, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 35,        and    -   b) as heavy chain of the second full length antibody connected        to the light chain of the second full length antibody via a        peptide linker the amino acid sequence of SEQ ID NO: 34.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 36, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 38,        and    -   b) as heavy chain of the second full length antibody connected        to the light chain of the second full length antibody via a        peptide linker the amino acid sequence of SEQ ID NO: 37.

In one embodiment the antibody heavy chain variable domain (VH) and theantibody light chain variable domain (VL) of the heavy and light chainof the second full length antibody are disulfide stabilized byintroduction of a disulfide bond between the following positions: heavychain variable domain position 44 to light chain variable domainposition 100 (numbering always according to EU index of Kabat (Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed.,Public Health Service, National Institutes of Health, Bethesda, Md.(1991)). Such further disulfide stabilization is achieved by theintroduction of a disulfide bond between the variable domains VH and VLof the second full length antibody heavy and light chain. Techniques tointroduce unnatural disulfide bridges for stabilization are describede.g. in WO 94/029350, Rajagopal, V., et al, Prot. Engin. 10 (1997)1453-59; Kobayashi et al., Nuclear Medicine & Biology 25 (1998) 387-393;or Schmidt, M., et al., Oncogene 18 (1999) 1711-1721.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 33, of SEQ ID NO: 34, and of SEQ ID NO: 35.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 36, of SEQ ID NO: 37, and of SEQ ID NO: 38.

In one embodiment the CH3 domains of the bispecific, bivalent antibodyaccording to the invention is altered by the “knob-into-holes”technology which is described in detail with several examples in e.g. WO96/027011, Ridgway J. B., et al., Protein Eng 9 (1996) 617-621; andMerchant, A. M., et al., Nat Biotechnol 16 (1998) 677-681. In thismethod the interaction surfaces of the two CH3 domains are altered toincrease the heterodimerisation of both heavy chains containing thesetwo CH3 domains. Each of the two CH3 domains (of the two heavy chains)can be the “knob”, while the other is the “hole”. The introduction of adisulfide bridge stabilizes the heterodimers (Merchant, A.M, et al.,Nature Biotech 16 (1998) 677-681; Atwell, S., et al. J. Mol. Biol. 270(1997) 26-35) and increases the yield.

In a preferred aspect of the invention all bispecific antibodiesaccording to the invention are characterized in that

the CH3 domain of one heavy chain and the CH3 domain of the other heavychain each meet at an interface which comprises an original interfacebetween the antibody CH3 domains;

wherein said interface is altered to promote the formation of thebispecific antibody, wherein the alteration is characterized in that:

a) the CH3 domain of one heavy chain is altered,

so that within the original interface the CH3 domain of one heavy chainthat meets the original interface of the CH3 domain of the other heavychain within the bispecific antibody,

an amino acid residue is replaced with an amino acid residue having alarger side chain volume, thereby generating a protuberance within theinterface of the CH3 domain of one heavy chain which is positionable ina cavity within the interface of the CH3 domain of the other heavy chain

and

b) the CH3 domain of the other heavy chain is altered,

so that within the original interface of the second CH3 domain thatmeets the original interface of the first CH3 domain within thebispecific antibody

an amino acid residue is replaced with an amino acid residue having asmaller side chain volume, thereby generating a cavity within theinterface of the second CH3 domain within which a protuberance withinthe interface of the first CH3 domain is positionable.

Thus the antibody according to invention is preferably characterized inthat

-   -   the CH3 domain of the heavy chain of the full length antibody        of a) and the CH3 domain of the heavy chain of the full length        antibody of b) each meet at an interface which comprises an        alteration in the original interface between the antibody CH3        domains;    -   wherein i) in the CH3 domain of one heavy chain    -   an amino acid residue is replaced with an amino acid residue        having a larger side chain volume, thereby generating a        protuberance within the interface of the CH3 domain of one heavy        chain which is positionable in a cavity within the interface of        the CH3 domain of the other heavy chain    -   and wherein    -   ii) in the CH3 domain of the other heavy chain    -   an amino acid residue is replaced with an amino acid residue        having a smaller side chain volume, thereby generating a cavity        within the interface of the second CH3 domain within which a        protuberance within the interface of the first CH3 domain is        positionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), valine (V).

In one aspect of the invention both CH3 domains are further altered bythe introduction of cysteine (C) as amino acid in the correspondingpositions of each CH3 domain such that a disulfide bridge between bothCH3 domains can be formed.

In one embodiment, the bispecific antibody comprises a T366W mutation inthe CH3 domain of the “knobs chain” and T366S, L368A, Y407V mutations inthe CH3 domain of the “hole chain”. An additional interchain disulfidebridge between the CH3 domains can also be used (Merchant, A. M, et al.,Nature Biotech 16 (1998) 677-681) e.g. by introducing a Y349C mutationinto the CH3 domain of the “knobs chain” and a E356C mutation or a S354Cmutation into the CH3 domain of the “hole chain”.

In another embodiment, the bispecific antibody according to theinvention comprises Y349C, T366W mutations in one of the two CH3 domainsand E356C, T366S, L368A, Y407V mutations in the other of the two CH3domains. In a another preferred embodiment the bispecific antibodycomprises Y349C, T366W mutations in one of the two CH3 domains andS354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains(the additional Y349C mutation in one CH3 domain and the additionalE356C or S354C mutation in the other CH3 domain forming a interchaindisulfide bridge) (numbering always according to EU index of Kabat(Kabat, E. A., et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991)). But also other knobs-in-holes technologies as described by

EP 1 870 459 Al, can be used alternatively or additionally. Thus anotherexample for the bispecific antibody are R409D; K370E mutations in theCH3 domain of the “knobs chain” and D399K; E357K mutations in the CH3domain of the “hole chain” (numbering always according to EU index ofKabat (Kabat, E. A., et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991)).

In another embodiment the bispecific antibody comprises a T366W mutationin the CH3 domain of the “knobs chain” and T366S, L368A, Y407V mutationsin the CH3 domain of the “hole chain” and additionally R409D; K370Emutations in the CH3 domain of the “knobs chain” and D399K; E357Kmutations in the CH3 domain of the “hole chain”.

In another embodiment the bispecific antibody comprises Y349C, T366Wmutations in one of the two CH3 domains and S354C, T366S, L368A, Y407Vmutations in the other of the two CH3 domains or said trivalent,bispecific antibody comprises Y349C, T366W mutations in one of the twoCH3 domains and S354C, T366S, L368A, Y407V mutations in the other of thetwo CH3 domains and additionally R409D; K370E mutations in the CH3domain of the “knobs chain” and D399K; E357K mutations in the CH3 domainof the “hole chain”.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in having one or more of the followingproperties (determined in assays as described in Example 6

-   -   shows a lower serum concentration compared to corresponding        bispecific antibody without the mutations described under iii)        (96 hours after intravitreal application in mice, which are        mouse FcRn deficient, but hemizygous transgenic for human FcRn);    -   shows a similar (factor 0.8 to 1.2) concentration in whole right        eye lysates compared to corresponding bispecific antibody        without the mutations described under iii) (in mice, which are        mouse FcRn deficient, but hemizygous transgenic for human FcRn,        96 hours after intravitreal application in the right eye).

In one embodiment the bispecific, bivalent antibody is characterized incomprising

a first antigen-binding site that specifically binds to human VEGF and asecond antigen-binding site that specifically binds to human ANG-2,characterized in that

-   -   i) said first antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 7, and as light chain        variable domain (VL) the SEQ ID NO: 8; and    -   ii) said second antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 15, and as light chain        variable domain (VL) the SEQ ID NO: 16; and    -   iii) the bispecific antibody comprises a constant heavy chain        region of IgG1 or IgG4 subclass (derived from human origin and)        comprising the mutations I253A, H310A, and H435A (numbering        according to EU Index of Kabat) and having one or more of the        following properties (determined in assays as described in        Example 6    -   shows a lower serum concentration compared to corresponding        bispecific antibody without the mutations described under iii)        (96 hours after intravitreal application in mice, which are        mouse FcRn deficient, but hemizygous transgenic for human FcRn);    -   shows a similar (factor 0.8 to 1.2) concentration in whole right        eye lysates compared to corresponding bispecific antibody        without the mutations described under iii) (in mice, which are        mouse FcRn deficient, but hemizygous transgenic for human FcRn,        96 hours after intravitreal application in the right eye).

In one embodiment the bispecific antibody is characterized in comprisinga first antigen-binding site that specifically binds to human VEGF and asecond antigen-binding site that specifically binds to human ANG-2,characterized in that

-   -   i) said first antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 7 with 1, 2 or 3 amino acid        residue substitutions, and as light chain variable domain (VL)        the SEQ ID NO: 8 with 1, 2 or 3 amino acid residue        substitutions; and    -   ii) said second antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 15 with 1, 2 or 3 amino acid        residue substitutions, and as light chain variable domain (VL)        the SEQ ID NO: with 1, 2 or 3 amino acid residue substitutions;        and    -   iii) the bispecific antibody comprises a constant heavy chain        region of IgG1 or IgG4 subclass (derived from human origin and)        comprising the mutations I253A, H310A, and H435A (numbering        according to EU Index of Kabat)

and having one or more of the following properties (determined in assaysas described in Example 6

-   -   shows a lower serum concentration compared to corresponding        bispecific antibody without the mutations described under iii)        (96 hours after intravitreal application in mice, which are        mouse FcRn deficient, but hemizygous transgenic for human FcRn);    -   shows a similar (factor 0.8 to 1.2) concentration in whole right        eye lysates compared to corresponding bispecific antibody        without the mutations described under iii) (in mice, which are        mouse FcRn deficient, but hemizygous transgenic for human FcRn,        96 hours after intravitreal application in the right eye).

As used herein, “antibody” refers to a binding protein that comprisesantigen-binding sites. The terms “binding site” or “antigen-bindingsite” as used herein denotes the region(s) of an antibody molecule towhich a ligand actually binds. The term “antigen-binding site” comprisesan antibody heavy chain variable domains (VH) and an antibody lightchain variable domains (VL) (pair of VH/VL)).

Antibody specificity refers to selective recognition of the antibody fora particular epitope of an antigen. Natural antibodies, for example, aremonospecific.

“Bispecific antibodies” according to the invention are antibodies whichhave two different antigen-binding specificities. Antibodies of thepresent invention are specific for two different antigens, VEGF as firstantigen and ANG-2 as second antigen.

The term “monospecific” antibody as used herein denotes an antibody thathas one or more binding sites each of which bind to the same epitope ofthe same antigen.

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites in an antibody molecule.As such, the terms “bivalent”, “tetravalent”, and “hexavalent” denotethe presence of two binding site, four binding sites, and six bindingsites, respectively, in an antibody molecule. The bispecific antibodiesaccording to the invention are preferably “bivalent”.

The term “VEGF” as used herein refers to human vascular endothelialgrowth factor (VEGF/VEGF-A,) the 165-amino acid human vascularendothelial cell growth factor (amino acid 27-191 of precursor sequenceof human VEGF165: SEQ

ID NO: 17; amino acids 1-26 represent the signal peptide), and related121, 189, and 206 vascular endothelial cell growth factor isoforms, asdescribed by Leung, D. W., et al., Science 246 (1989) 1306-9; Houck etal., Mol. Endocrin. 5 (1991) 1806 -1814; Keck, P. J., et al., Science246 (1989) 1309-12 and Connolly, D. T., et al., J. Biol. Chem. 264(1989) 20017-24; together with the naturally occurring allelic andprocessed forms of those growth factors. VEGF is involved in theregulation of normal and abnormal angiogenesis and neovascularizationassociated with tumors and intraocular disorders (Ferrara, N., et al.,Endocr. Rev. 18 (1997) 4-25; Berkman, R. A.,et al., J. Clin. Invest. 91(1993) 153-159; Brown, L. F., et al., Human Pathol. 26 (1995) 86-91;Brown, L. F., et al., Cancer Res. 53 (1993) 4727-4735; Mattern, J., etal., Brit. J. Cancer. 73 (1996) 931-934; and Dvorak, H. F., et al., Am.J. Pathol. 146 (1995) 1029-1039). VEGF is a homodimeric glycoproteinthat has been isolated from several sources and includes severalisoforms. VEGF shows highly specific mitogenic activity for endothelialcells.

The term “ANG-2” as used herein refers to human angiopoietin-2 (ANG-2)(alternatively abbreviated with ANGPT2 or ANG2) (SEQ ID NO: 18) which isdescribed e.g. in Maisonpierre, P. C., et al, Science 277 (1997) 55-60and Cheung, A. H., et al., Genomics 48 (1998) 389-91. Theangiopoietins-1 (SEQ ID NO: 19) and −2 were discovered as ligands forthe Ties, a family of tyrosine kinases that is selectively expressedwithin the vascular endothelium (Yancopoulos, G. D., et al., Nature 407(2000) 242-48). There are now four definitive members of theangiopoietin family. Angiopoietin-3 and -4 (Ang-3 and Ang-4) mayrepresent widely diverged counterparts of the same gene locus in mouseand man (Kim, I., et al., FEBS Let, 443 (1999) 353-56; Kim, I., et al.,J Biol Chem 274 (1999) 26523-28). ANG-1 and ANG-2 were originallyidentified in tissue culture experiments as agonist and antagonist,respectively (see for ANG-1: Davis, S., et al., Cell 87 (1996) 1161-69;and for ANG-2: Maisonpierre, P. C., et al., Science 277 (1997) 55-60).All of the known angiopoietins bind primarily to Tie2 (SEQ ID NO: 20),and both Ang-1 and -2 bind to Tie2 with an affinity of 3 nM (Kd)(Maisonpierre, P. C., et al., Science 277 (1997) 55-60).

An antigen-binding sites of the bispecific antibody of the inventioncontain six complementarity determining regions (CDRs) which contributein varying degrees to the affinity of the binding site for antigen.There are three heavy chain variable domain CDRs (CDRH1, CDRH2 andCDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2 andCDRL3). The extent of CDR and framework regions (FRs) is determined bycomparison to a compiled database of amino acid sequences in which thoseregions have been defined according to variability among the sequences.

The antibodies of the invention comprise immunoglobulin constant regionsderived from human origin of one or more immunoglobulin classes, whereinsuch. immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE classesand, in the case of IgG and IgA, their subclasses, especially IgG1 andIgG4.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The term “chimeric antibody” refers to an antibody comprising a variableregion, i.e., binding region, from one source or species and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Chimeric antibodiescomprising a murine variable region and a human constant region arepreferred. Other preferred forms of “chimeric antibodies” encompassed bythe present invention are those in which the constant region has beenmodified or changed from that of the original antibody to generate theproperties according to the invention, especially in regard to C1qbinding and/or Fc receptor (FcR) binding. Such chimeric antibodies arealso referred to as “class-switched antibodies.”. Chimeric antibodiesare the product of expressed immunoglobulin genes comprising DNAsegments encoding immunoglobulin variable regions and DNA segmentsencoding immunoglobulin constant regions. Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques are well known in the art. See, e.g., Morrison, S. L., etal., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. No.5,202,238 and U.S. Pat. No. 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the antigensnoted above for chimeric antibodies. Other forms of “humanizedantibodies” encompassed by the present invention are those in which theconstant region has been additionally modified or changed from that ofthe original antibody to generate the properties according to theinvention, especially in regard to C1q binding and/or Fc receptor (FcR)binding.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in thestate of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin.Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced intransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire or a selection of human antibodies in theabsence of endogenous immunoglobulin production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge(see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;Brueggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodiescan also be produced in phage display libraries (Hoogenboom, H. R., andWinter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J.Mol. Biol. 222 (1991) 581-597). The techniques of Cole, A., et al. andBoerner, P., et al. are also available for the preparation of humanmonoclonal antibodies (Cole, A., et al., Monoclonal Antibodies andCancer Therapy, Liss, A. L., p. 77 (1985); and Boerner, P., et al., J.Immunol. 147 (1991) 86-95). As already mentioned for chimeric andhumanized antibodies according to the invention the term “humanantibody” as used herein also comprises such antibodies which aremodified in the constant region to generate the properties according tothe invention, especially in regard to C1q binding and/or FcR binding,e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. fromIgG1 to IgG4 and/or IgG1/IgG4 mutation).

The term “recombinant antibody”, as used herein, is intended to includeall human antibodies that are prepared, expressed, created or isolatedby recombinant means, such as antibodies isolated from a host cell suchas a NS0 or CHO cell or from an animal (e.g. a mouse) that is transgenicfor human immunoglobulin genes or antibodies expressed using arecombinant expression vector transfected into a host cell. Suchrecombinant antibodies have variable and constant regions in arearranged form. The recombinant antibodies according to the inventionhave been subjected to in vivo somatic hypermutation. Thus, the aminoacid sequences of the VH and VL regions of the recombinant antibodiesare sequences that, while derived from and related to human germ line VHand VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

The “variable domain” (variable domain of a light chain (VL), variabledomain of a heavy chain (VH) as used herein denotes each of the pair oflight and heavy chains which is involved directly in binding theantibody to the antigen. The domains of variable human light and heavychains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions” (or complementarity determiningregions, CDRs). The framework regions adopt a β-sheet conformation andthe CDRs may form loops connecting the β-sheet structure. The CDRs ineach chain are held in their three-dimensional structure by theframework regions and form together with the CDRs from the other chainthe antigen binding site. The antibody heavy and light chain CDR3regions play a particularly important role in the bindingspecificity/affinity of the antibodies according to the invention andtherefore provide a further object of the invention.

The terms “hypervariable region” or “antigen-binding portion of anantibody” when used herein refer to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from the “complementaritydetermining regions” or “CDRs”. “Framework” or “FR” regions are thosevariable domain regions other than the hypervariable region residues asherein defined. Therefore, the light and heavy chains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. CDRs on each chain are separated by such framework aminoacids. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding. CDR and FR regions are determinedaccording to the standard definition of Kabat, E. A., et al., Sequencesof Proteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991).

As used herein, the term “binding” or “specifically binding” refers tothe binding of the antibody to an epitope of the antigen (either humanVEGF or human ANG-2) in an in vitro assay, preferably in an plasmonresonance assay (BlAcore, GE-Healthcare Uppsala, Sweden with purifiedwild-type antigen. The affinity of the binding is defined by the termska (rate constant for the association of the antibody from theantibody/antigen complex), k_(D) (dissociation constant), and K_(D)(k_(D)/ka). In one embodiment binding or specifically binding means abinding affinity (K_(D)) of 10⁻⁸ mol/l or less, in one embodiment 10⁻⁹ Mto 10⁻¹³ mol/l.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminant include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody.

In certain embodiments, an antibody is said to specifically bind anantigen when it preferentially recognizes its target antigen in acomplex mixture of proteins and/or macromolecules.

The term “full length antibody” denotes an antibody consisting of two“full length antibody heavy chains” and two “full length antibody lightchains”. A “full length antibody heavy chain” is a polypeptideconsisting in N-terminal to C-terminal direction of an antibody heavychain variable domain (VH), an antibody constant heavy chain domain 1(CH1), an antibody hinge region (HR), an antibody heavy chain constantdomain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3),abbreviated as VH-CH1-HR-CH2-CH3; and optionally an antibody heavy chainconstant domain 4 (CH4) in case of an antibody of the subclass IgE.Preferably the “full length antibody heavy chain” is a polypeptideconsisting in N-terminal to C-terminal direction of VH, CH1, HR, CH2 andCH3. A “full length antibody light chain” is a polypeptide consisting inN-terminal to C-terminal direction of an antibody light chain variabledomain (VL), and an antibody light chain constant domain (CL),abbreviated as VL-CL. The antibody light chain constant domain (CL) canbe κ (kappa) or λ (lambda). The two full length antibody chains arelinked together via inter-polypeptide disulfide bonds between the CLdomain and the CH1 domain and between the hinge regions of the fulllength antibody heavy chains. Examples of typical full length antibodiesare natural antibodies like IgG (e.g. IgG 1 and IgG2), IgM, IgA, IgD,and IgE. The full length antibodies according to the invention can befrom a single species e.g. human, or they can be chimerized or humanizedantibodies. The full length antibodies according to the inventioncomprise two antigen binding sites each formed by a pair of VH and VL,which both specifically bind to the same antigen. The C-terminus of theheavy or light chain of said full length antibody denotes the last aminoacid at the C-terminus of said heavy or light chain. The N-terminus ofthe heavy or light chain of said full length antibody denotes the lastamino acid at the N-terminus of said heavy or light chain.

The term “peptide linker” as used within the invention denotes a peptidewith amino acid sequences, which is preferably of synthetic origin.These peptides according to invention are used to connect the C-terminusof the light chain to the N-terminus of heavy chain of the second fulllength antibody (that specifically binds to a second antigen) via apeptide linker. The peptide linker within the second full lengthantibody heavy and light chain is a peptide with an amino acid sequencewith a length of at least 30 amino acids, preferably with a length of 32to 50 amino acids. In one the peptide linker is a peptide with an aminoacid sequence with a length of 32 to 40 amino acids. In one embodimentsaid linker is (GxS)n with G=glycine, S=serine, (x=3, n=8, 9 or 10 andm=0, 1, 2 or 3) or (x=4 and n=6, 7 or 8 and m=0, 1, 2 or 3), preferablywith x=4, n=6 or 7 and m=0, 1, 2 or 3, more preferably with x=4, n=7 andm=2. In one embodiment said linker is (G₄S)₆G₂.

The term “constant region” as used within the current applicationsdenotes the sum of the domains of an antibody other than the variableregion. The constant region is not involved directly in binding of anantigen, but exhibits various effector functions. Depending on the aminoacid sequence of the constant region of their heavy chains, antibodiesare divided in the classes: IgA, IgD, IgE, IgG and IgM, and several ofthese may be further divided into subclasses, such as IgG1, IgG2, IgG3,and IgG4, IgA1 and IgA2. The heavy chain constant regions thatcorrespond to the different classes of antibodies are called α, δ, ε, γ,and μ, respectively. The light chain constant regions which can be foundin all five antibody classes are called κ (kappa) and λ (lambda).

The terms “constant region derived from human origin” or “ humanconstant region” as used in the current application denotes a constantheavy chain region of a human antibody of the subclass IgG1, IgG2, IgG3,or IgG4 and/or a constant light chain kappa or lambda region. Suchconstant regions are well known in the state of the art and e.g.described by Kabat, E. A., et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) (see also e.g. Johnson, G.,and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E. A., etal., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Within theapplication for the numbering of positions and mutations the EUnumbering system (EU Index) according to Kabat, E. A., et al., Sequencesof Proteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991) is used and referredto as “numbering according to EU Index of Kabat”.

In one embodiment the bispecific antibodies according to the inventionhave a constant region of human IgG1 subclass (derived from human IgG1subclass) .

In one embodiment the bispecific antibodies according to the inventionhave a constant region of human IgG4 subclass (derived from human IgG1subclass).

In one embodiment the bispecific antibody according to the invention isof human IgG1 subclass with mutations L234A (Leu234Ala), L235A(Leu235Ala) and P329G (Pro329Gly). Such antibody has a reduced FcRbinding (especially they show no more binding to FcRgammaI, FcRgammaIIand FcRgammaIII). This especially useful to reduce potential sideeffects like e.g. thrombosis (Meyer, T., et al., J. Thromb. Haemost. 7(2009) 171-81). In one embodiment the bispecific antibody according tothe invention is of human IgG4 subclass with mutations S228P(Ser228Pro), L235E (Leu235Glu) and P329G (Pro329Gly). Such antibodyshows reduced FcR binding as indicated above. While Pro329Ala mutationwhich was described already removes only two third of the FcgammaRIIIasandwich interaction, the Pro329Gly in the antibodies according to theinvention fully imparts binding of the Fc part to FcgammaRIII. This isespecially useful as the binding to FcgammaRIII is involved in ADCC(antibody—dependent cellular toxicity) which leads to cell death, whichmay be helpful in the treatment of cancer diseases, but which can causeserious side effect in the antibody based treatment of other vascular orimmunological diseases. So the antibodies according to the invention ofIgG1 subclass with mutations L234A, L235A and P329G and IgG4 subclasswith mutations S228P, L235E and P329G are especially useful, as theyboth show no more binding to FcRgammaI, FcRgammaII and FcRgammaIII.

The term “with (the) mutations AAA” as used herein refers the mutationsI253A (Ile253Ala), H310A (His310Ala), and H435A (His435Ala) in theconstant heavy chain region of IgG1 or IgG4, wherein the numbering isaccording to the EU Index of Kabat.

The term “with (the) mutations P329G LALA” as used herein refers to themutations L234A (Leu234Ala), L235A (Leu235Ala) and P329G (Pro329Gly) inthe constant heavy chain region of IgG1 subclass, wherein the numberingis according to the EU Index of Kabat. The term “with (the) mutationsSPLE” as used herein refers to the S228P (Ser228Pro) and L235E(Leu235Glu) the constant heavy chain region of IgG4 subclass, whereinthe numbering is according to the EU Index of Kabat. The term “with(the) mutations SPLE and P239G” as used herein refers to the S228P(Ser228Pro), L235E (Leu235Glu) and P329G (Pro329Gly) the constant heavychain region of IgG4 subclass, wherein the numbering is according to theEU Index of Kabat.

The antibody according to the invention is produced by recombinantmeans. Thus, one aspect of the current invention is a nucleic acidencoding the antibody according to the invention and a further aspect isa cell comprising said nucleic acid encoding an antibody according tothe invention. Methods for recombinant production are widely known inthe state of the art and comprise protein expression in prokaryotic andeukaryotic cells with subsequent isolation of the antibody and usuallypurification to a pharmaceutically acceptable purity. For the expressionof the antibodies as aforementioned in a host cell, nucleic acidsencoding the respective modified light and heavy chains are insertedinto expression vectors by standard methods. Expression is performed inappropriate prokaryotic or eukaryotic host cells like CHO cells, NSOcells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, orE.coli cells, and the antibody is recovered from the cells (supernatantor cells after lysis). General methods for recombinant production ofantibodies are well-known in the state of the art and described, forexample, in the review articles of Makrides, S. C., Protein Expr. Purif17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996)271-282; Kaufman, R. J., Mol. Biotechnol. 16 (2000) 151-160; Werner, R.G., Drug Res. 48 (1998) 870-880.

Accordingly one embodiment of the invention is a method for thepreparation of a bispecific antibody according to the invention,comprising the steps of

-   -   a) transforming a host cell with vectors comprising nucleic acid        molecules encoding said antibody;    -   b) culturing the host cell under conditions that allow synthesis        of said antibody molecule; and    -   c) recovering said antibody molecule from said culture.

In one embodiment the recovering step under c includes the use of alight chain constant domain specific capture reagent (which e.g.specific for the kappa or the lambda constant light chain, depending onwhether a kappa or a lambda light chain in the bispecific antibodyaccording to invention used). In one embodiment this light chainspecific capture reagent is used in in a bind-and-elute-mode). Examplesof such light chain constant domain specific capture reagents are e.g.KappaSelect™ and LambdaFabSelect™ from GE Healthcare/BAC, which arebased on a highly rigid agarose base matrix that allows high flow ratesand low back pressure at large scale. They feature a ligand that bindsto the constant region of the kappa or the lambda light chainrespectively (i.e. fragments lacking the constant region of the lightchain will not bind; FIG. 1). Both are therefore capable of bindingother target molecules containing the constant region of the lightchain, for example, IgG, IgA and IgM. The ligands are attached to thematrix via a long hydrophilic spacer arm to make it easily available forbinding to the target molecule. They are based on a single-chainantibody fragment that is screened for either human Ig kappa or lambda.

The bispecific antibodies are suitably separated from the culture mediumby conventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA and RNAencoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures. The hybridoma cells can serve as a sourceof such DNA and RNA. Once isolated, the DNA may be inserted intoexpression vectors, which are then transfected into host cells such asHEK 293 cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantmonoclonal antibodies in the host cells.

Amino acid sequence variants (or mutants) of the bispecific antibody areprepared by introducing appropriate nucleotide changes into the antibodyDNA, or by nucleotide synthesis. Such modifications can be performed,however, only in a very limited range. For example, the modifications donot alter the above mentioned antibody characteristics such as the IgGsubclass and antigen binding, but may improve the yield of therecombinant production, protein stability or facilitate thepurification.

The term “host cell” as used in the current application denotes any kindof cellular system which can be engineered to generate the antibodiesaccording to the current invention. In one embodiment HEK293 cells andCHO cells are used as host cells. As used herein, the expressions“cell,” “cell line,” and “cell culture” are used interchangeably and allsuch designations include progeny. Thus, the words “transformants” and“transformed cells” include the primary subject cell and culturesderived therefrom without regard for the number of transfers. It is alsounderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological activity as screened for inthe originally transformed cell are included.

Expression in NSO cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

A nucleic acid is “operably linked” when it is placed in a functionalrelationship with another nucleic acid sequence. For example, DNA for apre-sequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a pre-protein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Purification of antibodies is performed in order to eliminate cellularcomponents or other contaminants, e.g. other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, agarose gel electrophoresis, and otherswell known in the art. See Ausubel, F., et al., ed. Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York(1987). Different methods are well established and widespread used forprotein purification, such as affinity chromatography with microbialproteins (e.g. protein A or protein G affinity chromatography), ionexchange chromatography (e.g. cation exchange (carboxymethyl resins),anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilicadsorption (e.g. with beta-mercaptoethanol and other SH ligands),hydrophobic interaction or aromatic adsorption chromatography (e.g. withphenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid),metal chelate affinity chromatography (e.g. with Ni(II)- andCu(II)-affinity material), size exclusion chromatography, andelectrophoretical methods (such as gel electrophoresis, capillaryelectrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75(1998) 93-102).

The bispecific, bivalent antibodies according to the invention showbenefits for human patients in need of a VEGF and ANG-2 targetingtherapy.

The bivalent bispecific against human VEGF and human ANG-2 according tothe current invention may have a valuable efficacy/safety profile andmay provide benefits for a patient in the need of an anti-VEGF andanti-ANG-2 therapy.

One aspect of the invention is a pharmaceutical composition comprisingan antibody according to the invention. Another aspect of the inventionis the use of an antibody according to the invention for the manufactureof a pharmaceutical composition. A further aspect of the invention is amethod for the manufacture of a pharmaceutical composition comprising anantibody according to the invention. In another aspect, the presentinvention provides a composition, e.g. a pharmaceutical composition,containing an antibody according to the present invention, formulatedtogether with a pharmaceutical carrier.

As used herein, “pharmaceutical carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable foradministration administered to the subject via a local route. Forexample, the antibody or its composition can be administered to thesubject by intraocular application e.g. by intraocular injection such asintravitreal injection. This can be performed in accordance withstandard procedures known in the art. See, e.g., Ritter et al., J. Clin.Invest. 116 (2006) 3266-76; Russelakis-Carneiro et al., Neuropathol.Appl. Neurobiol. 25 (1999) 196-206; and Wray et al., Arch. Neurol. 33(1976) 183-5.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. To administer a compound of the invention bycertain routes of administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation. For example, the compound may be administered to asubject in an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Pharmaceutical carriers include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.

Many possible modes of delivery can be used, including, but not limitedto intraocular application or topical application. In one embodiment theapplication is intraocular and includes, but is not limited to,subconjunctival injection, intracanieral injection, injection into theanterior chamber via the termporal limbus, intrastromal injection,intracorneal injection, subretinal injection, aqueous humor injection,subtenon injection or sustained delivery device, intravitreal injection(e.g., front, mid or back vitreal injection). In one embodiment theapplication is topical and includes, but is not limited to eye drops tothe cornea.

In one embodiment the bispecific antibody or pharmaceutical compositionaccording to the invention is administered via intravitreal application,e.g. via intravitreal injection. This can be performed in accordancewith standard procedures known in the art. See, e.g., Ritter et al., J.Clin. Invest. 116 (2006) 3266-76; Russelakis-Carneiro et al.,Neuropathol. Appl. Neurobiol. 25 (1999) 196-206; and Wray et al., Arch.Neurol. 33 (1976) 183-5.

In some embodiments, therapeutic kits of the invention can contain oneor more doses of a bispecific antibody present in a pharmaceuticalcomposition described herein, a suitable device for intravitrealinjection of the pharmaceutical composition, and an instructiondetailing suitable subjects and protocols for carrying out theinjection. In these embodiments, the compositions are typicallyadministered to the subject in need of treatment via intravitrealinjection. This can be performed in accordance with standard proceduresknown in the art. See, e.g., Ritter et al., J. Clin. Invest. 116 (2006)3266-76; Russelakis-Carneiro et al., Neuropathol. Appl. Neurobiol. 25(1999) 196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-5.

The compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carrierpreferably is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size in the case ofdispersion and by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the composition.

The composition can comprise an ophthalmic depot formulation comprisingan active agent for subconjunctival administration. The ophthalmic depotformulation comprises microparticles of essentially pure active agent,e.g., the bispecific antibody according to the invention. Themicroparticles comprising the bispecific antibody according to theinvention can be embedded in a biocompatible pharmaceutically acceptablepolymer or a lipid encapsulating agent. The depot formulations may beadapted to release all of substantially all the active material over anextended period of time. The polymer or lipid matrix, if present, may beadapted to degrade sufficiently to be transported from the site ofadministration after release of all or substantially all the activeagent. The depot formulation can be liquid formulation, comprising apharmaceutical acceptable polymer and a dissolved or dispersed activeagent. Upon injection, the polymer forms a depot at the injections site,e.g. by gelifying or precipitating.

Another aspect of the invention is the bispecific antibody according tothe invention for use in the treatment of ocular vascular diseases.

One embodiment of the invention is the bispecific antibody according tothe invention for use in the treatment of ocular vascular diseases.

Another aspect of the invention is said pharmaceutical composition foruse in the treatment of ocular vascular diseases.

Another aspect of the invention is the use of an antibody according tothe invention for the manufacture of a medicament for the treatment ofocular vascular disease.

Another aspect of the invention is method of treatment of patientsuffering from ocular vascular diseases by administering an antibodyaccording to the invention to a patient in the need of such treatment.

The terms “ocular vascular disease” and “vascular eye disease” are useinter changeable herein and include, but are not limited to intraocularneovascular syndromes such as diabetic retinopathy, diabetic macularedema—retinopathy of prematurity, neovascular glaucoma, retinal veinocclusions, central retinal vein occlusions, macular degeneration,age-related macular degeneration, retinitis pigmentosa, retinalangiomatous proliferation, macular telangectasia, ischemic retinopathy,iris neovascularization, intraocular neovascularization, cornealneovascularization, retinal neovascularization, choroidalneovascularization, and retinal degeneration. (Garner, A., Vasculardiseases, In: Pathobiology of ocular disease, A dynamic approach,Garner, A., and Klintworth, G. K., (eds.), 2nd edition, Marcel Dekker,New York (1994), pp. 1625-1710). As used herein, ocular vasculardisorder refers to any pathological conditions characterized by alteredor unregulated proliferation and invasion of new blood vessels into thestructures of ocular tissues such as the retina or cornea. In oneembodiment the ocular vascular disease is selected from the groupconsisting of: wet age-related macular degeneration (wet AMD), dryage-related macular degeneration (dry AMD), diabetic macular edema(DME), cystoid macular edema (CME), non-proliferative diabeticretinopathy (NPDR), proliferative diabetic retinopathy (PDR), cystoidmacular edema, vasculitis (e.g. central retinal vein occlusion),papilloedema, retinitis, conjunctivitis, uveitis, choroiditis,multifocal choroiditis, ocular histoplasmosis, blepharitis, dry eye(Sjögren's disease) and other ophthalmic diseases wherein the eyedisease or disorder is associated with ocular neovascularization,vascular leakage, and/or retinal edema. So the bispecific antibodiesaccording to the invention are useful in the prevention and treatment ofwet AMD, dry AMD, CME, DME, NPDR, PDR, blepharitis, dry eye and uveitis,also preferably wet AMD, dry AMD, blepharitis, and dry eye, alsopreferably CME, DME, NPDR and PDR, also preferably blepharitis, and dryeye, in particular wet AMD and dry AMD, and also particularly wet AMD.In some embodiments, the ocular disease is selected from the groupconsisting of wet age-related macular degeneration (wet AMD), macularedema, retinal vein occlusions, retinopathy of prematurity, and diabeticretinopathy.

Other diseases associated with corneal neovascularization include, butare not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency,contact lens overwear, atopic keratitis, superior limbic keratitis,pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis,syphilis, Mycobacteria infections, lipid degeneration, chemical burns,bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpeszoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer,Terrien's marginal degeneration, mariginal keratolysis, rheumatoidarthritis, systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis,Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, andcorneal graph rejection.

Diseases associated with retinal/choroidal neovascularization include,but are not limited to, diabetic retinopathy, macular degeneration,sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagetsdisease, vein occlusion, artery occlusion, carotid obstructive disease,chronic uveitis/vitritis, mycobacterial infections, Lyme's disease,systemic lupus erythematosis, retinopathy of prematurity, retinitispigmentosa, retina edema (including macular edema), Eales disease,Bechets disease, infections causing a retinitis or choroiditis, presumedocular histoplasmosis, Bests disease, myopia, optic pits, Stargartsdisease, pars planitis, chronic retinal detachment, hyperviscositysyndromes, toxoplasmosis, trauma and post-laser complications. Otherdiseases include, but are not limited to, diseases associated withrubeosis (neovascularization of the angle) and diseases caused by theabnormal proliferation of fibrovascular or fibrous tissue including allforms of proliferative vitreoretinopathy.

Retinopathy of prematurity (ROP) is a disease of the eye that affectsprematurely born babies. It is thought to be caused by disorganizedgrowth of retinal blood vessels which may result in scarring and retinaldetachment. ROP can be mild and may resolve spontaneously, but may leadto blindness in serious cases. As such, all preterm babies are at riskfor ROP, and very low birth weight is an additional risk factor. Bothoxygen toxicity and relative hypoxia can contribute to the developmentof ROP.

Macular degeneration is a medical condition predominantly found inelderly adults in which the center of the inner lining of the eye, knownas the macula area of the retina, suffers thinning, atrophy, and in somecases, bleeding. This can result in loss of central vision, whichentails inability to see fine details, to read, or to recognize faces.According to the American Academy of Ophthalmology, it is the leadingcause of central vision loss (blindness) in the United States today forthose over the age of fifty years. Although some macular dystrophiesthat affect younger individuals are sometimes referred to as maculardegeneration, the term generally refers to age-related maculardegeneration (AMD or ARMD).

Age-related macular degeneration begins with characteristic yellowdeposits in the macula (central area of the retina which providesdetailed central vision, called fovea) called drusen between the retinalpigment epithelium and the underlying choroid. Most people with theseearly changes (referred to as age-related maculopathy) have good vision.People with drusen can go on to develop advanced AMD. The risk isconsiderably higher when the drusen are large and numerous andassociated with disturbance in the pigmented cell layer under themacula. Large and soft drusen are related to elevated cholesteroldeposits and may respond to cholesterol lowering agents or the RheoProcedure.

Advanced AMD, which is responsible for profound vision loss, has twoforms: dry and wet. Central geographic atrophy, the dry form of advancedAMD, results from atrophy to the retinal pigment epithelial layer belowthe retina, which causes vision loss through loss of photoreceptors(rods and cones) in the central part of the eye. While no treatment isavailable for this condition, vitamin supplements with high doses ofantioxidants, lutein and zeaxanthin, have been demonstrated by theNational Eye Institute and others to slow the progression of dry maculardegeneration and in some patients, improve visual acuity.

Retinitis pigmentosa (RP) is a group of genetic eye conditions. In theprogression of symptoms for RP, night blindness generally precedestunnel vision by years or even decades. Many people with RP do notbecome legally blind until their 40s or 50s and retain some sight alltheir life. Others go completely blind from RP, in some cases as earlyas childhood. Progression of RP is different in each case. RP is a typeof hereditary retinal dystrophy, a group of inherited disorders in whichabnormalities of the photoreceptors (rods and cones) or the retinalpigment epithelium (RPE) of the retina lead to progressive visual loss.Affected individuals first experience defective dark adaptation ornyctalopia (night blindness), followed by reduction of the peripheralvisual field (known as tunnel vision) and, sometimes, loss of centralvision late in the course of the disease.

Macular edema occurs when fluid and protein deposits collect on or underthe macula of the eye, a yellow central area of the retina, causing itto thicken and swell. The swelling may distort a person's centralvision, as the macula is near the center of the retina at the back ofthe eyeball. This area holds tightly packed cones that provide sharp,clear central vision to enable a person to see form, color, and detailthat is directly in the line of sight. Cystoid macular edema is a typeof macular edema that includes cyst formation.

Combination Therapies: In certain embodiments the bispecific antibody orpharmaceutical composition according to the invention is administeredalone (without an additional therapeutic agent) for the treatment of oneor more ocular vascular diseases described herein.

In other embodiments the bispecific antibody or pharmaceuticalcomposition according to the invention is administered in combinationwith one or more additional therapeutic agents or methods for thetreatment of one or more ocular vascular diseases described herein.

In other embodiments, the bispecific antibody or pharmaceuticalcomposition according to the invention is formulated in combination withone or more additional therapeutic agents and administered for thetreatment of one or more ocular vascular diseases described herein.

In certain embodiments, the combination treatments provided hereininclude administration the bispecific antibody or pharmaceuticalcomposition according to the invention is administered sequentially withone or more additional therapeutic agents for the treatment of one ormore ocular vascular diseases described herein.

The additional therapeutic agents include, but are not limited to,Tryptophanyl-tRNA synthetase (TrpRS), EyeOOl (Anti-VEGF PegylatedAptamer), squalamine, RETAANE® (anecortave acetate for depot suspension;Alcon, Inc.), Combretastatin A4 Prodrug (CA4P), MACUGEN®, MIFEPREX®(mifepristone-ru486), subtenon triamcinolone acetonide, intravitrealcrystalline triamcinolone acetonide, Prinomastat (AG3340—syntheticmatrix metalloproteinase inhibitor, Pfizer), fluocinolone acetonide(including fluocinolone intraocular implant, Bausch & Lomb/ControlDelivery Systems), VEGFR inhibitors (Sugen), VEGF-Trap(Regeneron/Aventis), VEGF receptor tyrosine kinase inhibitors such as4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(l-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787) and SU1 1248 (sunitinib), linomide, andinhibitors of integrin v.beta.3 function and angiostatin.

Other pharmaceutical therapies that can be sued used in combination thebispecific antibody or pharmaceutical composition according to theinvention is administered, include, but are not limited to, VISUDYNE®with use of a non-thermal laser, PKC 412, Endovion (NeuroSearch A/S),neurotrophic factors, including by way of example Glial DerivedNeurotrophic Factor and Ciliary Neurotrophic Factor, diatazem,dorzolamide, Phototrop, 9-cis-retinal, eye medication (including EchoTherapy) including phospholine iodide or echothiophate or carbonicanhydrase inhibitors, AE-941 (AEterna Laboratories, Inc.), Sirna-027(Sima Therapeutics, Inc.), pegaptanib (NeXstar Pharmaceuticals/GileadSciences), neurotrophins (including, by way of example only, NT-4/5,Genentech), Candy (Acuity Pharmaceuticals), INS-37217 (InspirePharmaceuticals), integrin antagonists (including those from Jerini AGand Abbott Laboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E(BioDiem Ltd.), thalidomide (as used, for example, by EntreMed, Inc.),cardiotrophin-1 (Genentech), 2-methoxyestradiol (Allergan/Oculex),DL-8234 (Toray Industries), NTC-200 (Neurotech), tetrathiomolybdate(University of Michigan), LYN-002 (Lynkeus Biotech), microalgal compound(Aquasearch/Albany, Mera Pharmaceuticals), D-9120 (Celltech Group pic),ATX-S10 (Hamamatsu Photonics), TGF-beta 2 (Genzyme/Celtrix), tyrosinekinase inhibitors (Allergan, SUGEN, Pfizer), NX-278-L (NeXstarPharmaceuticals/Gilead Sciences), Opt-24 (OPTIS France SA), retinal cellganglion neuroprotectants (Cogent Neurosciences), N-nitropyrazolederivatives (Texas A&M University System), KP-102 (KrenitskyPharmaceuticals), cyclosporin A, Timited retinal translocation”,photodynamic therapy, (including, by way of example only,receptor-targeted PDT, Bristol-Myers Squibb, Co.; porfimer sodium forinjection with PDT; verteporfin, QLT Inc.; rostaporfin with PDT,Miravent Medical Technologies; talaporfin sodium with PDT, NipponPetroleum; motexafin lutetium, Pharmacyclics, Inc.), antisenseoligonucleotides (including, by way of example, products tested byNovagali Pharma SA and ISIS-13650, Isis Pharmaceuticals), laserphotocoagulation, drusen lasering, macular hole surgery, maculartranslocation surgery, implantable miniature telescopes, Phi-MotionAngiography (also known as Micro-Laser Therapy and Feeder VesselTreatment), Proton Beam Therapy, microstimulation therapy, RetinalDetachment and Vitreous Surgery, Scleral Buckle, Submacular Surgery,Transpupillary Thermotherapy, Photosystem I therapy, use of RNAinterference (RNAi), extracorporeal rheopheresis (also known as membranedifferential filtration and Rheotherapy), microchip implantation, stemcell therapy, gene replacement therapy, ribozyme gene therapy (includinggene therapy for hypoxia response element, Oxford Biomedica; Lentipak,Genetix; PDEF gene therapy, GenVec), photoreceptor/retinal cellstransplantation (including transplantable retinal epithelial cells,Diacrin, Inc.; retinal cell transplant, Cell Genesys, Inc.), andacupuncture.

Any anti-angiogenic agent can be used in combination with the bispecificantibody or pharmaceutical composition according to the invention,including, bu not limited to, those listed by Carmeliet and Jain, 2000,Nature 407:249-257. In certain embodiments, the anti-angiogenic agent isanother VEGF antagonist or a VEGF receptor antagonist such as VEGFvariants, soluble VEGF receptor fragments, aptamers capable of blockingVEGF or VEGFR, neutralizing anti-VEGFR antibodies, low molecule weightinhibitors of VEGFR tyrosine kinases and any combinations thereof andthese include anti-VEGF aptamers (e.g. Pegaptanib), soluble recombinantdecoy receptors (e.g. VEGF Trap). In certain embodiments, theanti-angiogenic agent is include corticosteroids, angiostatic steroids,anecortave acetate, angiostatin, endostatin, small interfering RNA'sdecreasing expression of VEGFR or VEGF ligand, post-VEGFR blockade withtyrosine kinase inhibitors, MMP inhibitors, IGFBP3, SDF-1 blockers,PEDF, gamma-secretase, Delta-like ligand 4, integrin antagonists, HIF-1alpha blockade, protein kinase CK2 blockade, and inhibition of stem cell(i.e. endothelial progenitor cell) homing to the site ofneovascularization using vascular endothelial cadherin (CD-144) andstromal derived factor (SDF)-I antibodies. Small molecule RTK inhibitorstargeting VEGF receptors including PTK787 can also be used. Agents thathave activity against neovascularization that are not necessarilyanti-VEGF compounds can also be used and include anti-inflammatorydrugs, m-Tor inhibitors, rapamycin, everolismus, temsirolismus,cyclospohne, anti-TNF agents, anti-complement agents, and nonsteroidalantiinflammatory agents. Agents that are neuroprotective and canpotentially reduce the progression of dry macular degeneration can alsobe used, such as the class of drugs called the ‘neurosteroids.’ Theseinclude drugs such as dehydroepiandrosterone(DHEA)(Brand names:Prastera™ and Fidelin®), dehydroepiandrosterone sulfate, andpregnenolone sulfate. Any AMD (age-related macular degeneration)therapeutic agent can be used in combination with the bispecificantibody or pharmaceutical composition according to the invention,including but not limited to verteporfin in combination with PDT,pegaptanib sodium, zinc, or an antioxidant(s), alone or in anycombination.

The terms “subject” and “patient” are used interchangeably and refer tomammals such as human patients and non-human primates, as well asexperimental animals such as rabbits, rats, and mice, and other animals.Animals include all vertebrates, e.g., mammals and non-mammals, such asdogs, cats, sheeps, cows, pigs, rabbits, chickens, and etc. Preferredsubjects for practicing the therapeutic methods of the present inventionare human. Subjects in need of treatment include patients alreadysuffering from an ocular vascular disease or disorder as well as thoseprone to developing the disorder.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “transformation” as used herein refers to process of transferof a vectors/nucleic acid into a host cell. If cells without formidablecell wall barriers are used as host cells, transfection is carried oute.g. by the calcium phosphate precipitation method as described byGraham, F. L., van der Eb, A. J., Virology 52 (1973) 546-467. However,other methods for introducing DNA into cells such as by nuclearinjection or by protoplast fusion may also be used. If prokaryotic cellsor cells which contain substantial cell wall constructions are used,e.g. one method of transfection is calcium treatment using calciumchloride as described by Cohen, S. N., et al., PNAS. 69 (1972)2110-2114.

As used herein, “expression” refers to the process by which a nucleicacid is transcribed into mRNA and/or to the process by which thetranscribed mRNA (also referred to as transcript) is subsequently beingtranslated into peptides, polypeptides, or proteins. The transcripts andthe encoded polypeptides are collectively referred to as gene product.If the polynucleotide is derived from genomic DNA, expression in aeukaryotic cell may include splicing of the mRNA.

A “vector” is a nucleic acid molecule, in particular self-replicating,which transfers an inserted nucleic acid molecule into and/or betweenhost cells. The term includes vectors that function primarily forinsertion of DNA or RNA into a cell (e.g., chromosomal integration),replication of vectors that function primarily for the replication ofDNA or RNA, and expression vectors that function for transcriptionand/or translation of the DNA or RNA. Also included are vectors thatprovide more than one of the functions as described.

An “expression vector” is a polynucleotide which, when introduced intoan appropriate host cell, can be transcribed and translated into apolypeptide. An “expression system” usually refers to a suitable hostcell comprised of an expression vector that can function to yield adesired expression product.

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

Description of the Sequence Listing (Amino acid sequences) SEQ ID NO: 1heavy chain CDR3H, <VEGF> ranibizumab SEQ ID NO: 2 heavy chain CDR2H,<VEGF> ranibizumab SEQ ID NO: 3 heavy chain CDR1H, <VEGF> ranibizumabSEQ ID NO: 4 light chain CDR3L, <VEGF> ranibizumab SEQ ID NO: 5 lightchain CDR2L, <VEGF> ranibizumab SEQ ID NO: 6 light chain CDR1L, <VEGF>ranibizumab SEQ ID NO: 7 heavy chain variable domain VH, <VEGF>ranibizumab SEQ ID NO: 8 light chain variable domain VL, <VEGF>ranibizumab SEQ ID NO: 9 heavy chain CDR3H, <ANG-2> Ang2i_LC10 variantSEQ ID NO: 10 heavy chain CDR2H, <ANG-2> Ang2i_LC10 variant SEQ ID NO:11 heavy chain CDR1H, <ANG-2> Ang2i_LC10 variant SEQ ID NO: 12 lightchain CDR3L, <ANG-2> Ang2i_LC10 variant SEQ ID NO: 13 light chain CDR2L,<ANG-2> Ang2i_LC10 variant SEQ ID NO: 14 light chain CDR1L, <ANG-2>Ang2i_LC10 variant SEQ ID NO: 15 heavy chain variable domain VH, <ANG-2>Ang2i_LC10 variant SEQ ID NO: 16 light chain variable domain VL, <ANG-2>Ang2i_LC10 variant SEQ ID NO: 17 Human vascular endothelial growthfactor (VEGF); precursor sequence of human VEGF165 SEQ ID NO: 18 Humanangiopoietin-2 (ANG-2) SEQ ID NO: 19 Human angiopoietin-1 (ANG-1) SEQ IDNO: 20 Human Tie-2 receptor SEQ ID NO 21 Heavy chain 1 of <VEGF-ANG-2>CrossMAb IgG1 with AAA mutations (VEGFang2-0012) SEQ ID NO 22 Heavychain 2 of <VEGF-ANG-2> CrossMAb IgG1 with AAA mutations (VEGFang2-0012)SEQ ID NO 23 Light chain 1 of <VEGF-ANG-2> CrossMAb IgG1 with AAAmutations (VEGFang2-0012) SEQ ID NO 24 Light chain 2 of <VEGF-ANG-2>CrossMAb IgG1 with AAA mutations (VEGF-Ang2-0012) SEQ ID NO: 25 Heavychain 1 of <VEGF-ANG-2> CrossMAb IgG1 with AAA mutations and P329G LALAmutations (VEGFang2-0016) SEQ ID NO: 26 Heavy chain 2 of <VEGF-ANG-2>CrossMAb IgG1 with AAA mutations and P329G LALA mutations(VEGFang2-0016) SEQ ID NO: 27 Light chain 1 of <VEGF-ANG-2> CrossMAbIgG1 with AAA mutations and P329G LALA mutations (VEGFang2-0016) SEQ IDNO: 28 Light chain 2 of <VEGF-ANG-2> CrossMAb IgG1 with AAA mutationsand P329G LALA mutations (VEGFang2-0016) SEQ ID NO: 29 Heavy chain 1 of<VEGF-ANG-2> CrossMAb IgG4 with AAA mutations and with SPLE mutationsSEQ ID NO: 30 Heavy chain 2 of <VEGF-ANG-2> CrossMAb IgG4 with AAAmutations and with SPLE mutations SEQ ID NO: 31 Light chain 1 of<VEGF-ANG-2> CrossMAb IgG4 with AAA mutations and with SPLE mutationsSEQ ID NO: 32 Light chain 2 of <VEGF-ANG-2> CrossMAb IgG4 with AAAmutations and with SPLE mutations SEQ ID NO: 33 Heavy chain 1 of<VEGF-ANG-2> OAscFab IgG1 with AAA mutations SEQ ID NO: 34 Heavy chain 2of <VEGF-ANG-2> OAscFab IgG1 with AAA mutations SEQ ID NO: 35 Lightchain 1 of <VEGF-ANG-2> OAscFab IgG1 with AAA mutations SEQ ID NO: 36Heavy chain 1 of <VEGF-ANG-2> OAscFab IgG4 with AAA mutations and withSPLE mutations SEQ ID NO: 37 Heavy chain 2 of <VEGF-ANG-2> OAscFab IgG4with AAA mutations and with SPLE mutations SEQ ID NO: 38 Light chain 1of <VEGF-ANG-2> OAscFab IgG4 with AAA mutations and with SPLE mutationsSEQ ID NO: 39 Heavy chain 1 of <VEGF-ANG-2> CrossMAb IgG1 wild type(without AAA mutations) (VEGFang2-0201) SEQ ID NO: 40 Heavy chain 2 of<VEGF-ANG-2> CrossMAb IgG1 wild type (without AAA mutations)(VEGFang2-0201) SEQ ID NO: 41 Light chain 1 of <VEGF-ANG-2> CrossMAbIgG1 wild type (without AAA mutations) (VEGFang2-0201) SEQ ID NO: 42Light chain 2 of <VEGF-ANG-2> CrossMAb IgG1 wild type (without AAAmutations) (VEGFang2-0201) SEQ ID NO: 43 Heavy chain 1 of <VEGF-ANG-2>CrossMAb IgG1 with P329G LALA mutations only (without AAA mutations)(VEGFang2-0015) SEQ ID NO: 44 Heavy chain 2 of <VEGF-ANG-2> CrossMAbIgG1 with P329G LALA mutations only (without AAA mutations)(VEGFang2-0015) SEQ ID NO: 45 Light chain 1 of <VEGF-ANG-2> CrossMAbIgG1 with P329G LALA mutations only (without AAA mutations)(VEGFang2-0015) SEQ ID NO: 46 Light chain 2 of <VEGF-ANG-2> CrossMAbIgG1 with P329G LALA mutations only (without AAA mutations)(VEGFang2-0015) SEQ ID NO: 47 kappa light chain constant region SEQ IDNO: 48 lambda light chain constant region SEQ ID NO: 49 heavy chainconstant region derived from human IgG1 SEQ ID NO: 50 heavy chainconstant region derived from human IgG4

In the following, embodiments of the invention are listed:

-   1. A bispecific antibody comprising a first antigen-binding site    that specifically binds to human VEGF and a second antigen-binding    site that specifically binds to human ANG-2, wherein    -   i) said first antigen-binding site specifically binding to VEGF        comprises in the heavy chain variable domain a CDR3H region of        SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region        of SEQ ID NO:3, and in the light chain variable domain a CDR3L        region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a        CDR1L region of SEQ ID NO:6; and    -   ii) said second antigen-binding site specifically binding to        ANG-2 comprises in the heavy chain variable domain a CDR3H        region of SEQ ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a        CDR1H region of SEQ ID NO: 11, and in the light chain variable        domain a CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID        NO: 13, and a CDR1L region of SEQ ID NO: 14, and wherein    -   iii) the bispecific antibody comprises a constant heavy chain        region of human IgG1 or human IgG4 subclass (derived from human        origin and) comprising the mutations I253A, H310A, and H435A        (numbering according to EU Index of Kabat).-   2. The bispecific antibody according to embodiment 1, wherein    -   i) said first antigen-binding site specifically binding to VEGF        comprises as heavy chain variable domain VH an amino acid        sequence of SEQ ID NO: 7, and as light chain variable domain VL        an amino acid sequence of SEQ ID NO: 8, and    -   ii) said second antigen-binding site specifically binding to        ANG-2 comprises as heavy chain variable domain VH an amino acid        sequence of SEQ ID NO: 15, and as light chain variable domain VL        an amino acid sequence of SEQ ID NO: 16.-   3. The bispecific antibody according to any one of embodiments 1 to    2, wherein the constant heavy chain region under iii) is of IgG1    subclass.-   4. The bispecific antibody according to embodiment 3, wherein the    constant heavy chain region of IgG1 subclass further comprises the    mutations L234A, L235A and P329G (numbering according to EU Index of    Kabat).-   5. The bispecific antibody according to any one of embodiments 1 to    2, wherein the constant heavy chain region under iii) is of IgG4    subclass.-   6. The bispecific antibody according to embodiment 5, wherein the    constant heavy chain region of IgG4 subclass further comprises the    mutations S228P and L235E (numbering according to EU Index of    Kabat).-   7. The bispecific antibody according to embodiment 5, wherein the    constant heavy chain region of IgG4 subclass further comprises the    mutations S228P, L235E and P329G (numbering according to EU Index of    Kabat).-   8. A pharmaceutical composition comprising an antibody according to    any one of embodiments 1 to 7.-   9. The bispecific antibody according to any one of embodiments 1 to    7 for use in the treatment of ocular vascular diseases.-   10. Use of the bispecific antibody according to any one of    embodiments 1 to 7 for the manufacture of a medicament for the    treatment of ocular vascular diseases.-   11. The bispecific antibody according to any one of embodiments 9 or    10, wherein the antibody is administered via intravitreal    application.-   12. A method of treatment of patient suffering from ocular vascular    diseases by administering an antibody according to any one of    embodiments 1 to 7 to a patient in the need of such treatment.-   13. A nucleic acid encoding a bispecific antibody according to any    one of embodiments 1 to 7.-   14. Expression vector containing said nucleic acid according    embodiment 13 capable of expressing said nucleic acid in a    prokaryotic or eukaryotic host cell.-   15. A prokaryotic or eukaryotic host cell comprising a vector    according to embodiment 14.-   16. A method for the preparation of a bispecific antibody according    to embodiments 1 to 7    -   comprising the steps of    -   a) transforming a host cell with vectors comprising nucleic acid        molecules encoding said antibody;    -   b) culturing the host cell under conditions that allow synthesis        of said antibody molecule; and    -   c) recovering said antibody molecule from said culture.-   17. A bispecific antibody obtained by the method of embodiment 16.-   18. A bispecific, bivalent antibody comprising a first    antigen-binding site that specifically binds to human VEGF and a    second antigen-binding site that specifically binds to human ANG-2,    characterized in comprising the amino acid sequences of SEQ ID NO:    25, of SEQ ID NO: 26, of SEQ ID NO: 27, and of SEQ ID NO: 28.-   19. A bispecific, bivalent antibody comprising a first    antigen-binding site that specifically binds to human VEGF and a    second antigen-binding site that specifically binds to human ANG-2,    characterized in comprising the amino acid sequences of SEQ ID NO:    21, of SEQ ID NO: 22., of SEQ ID NO: 23., and of SEQ ID NO: 24.-   20. A bispecific, bivalent antibody comprising a first    antigen-binding site that specifically binds to human VEGF and a    second antigen-binding site that specifically binds to human ANG-2,    characterized in comprising the amino acid sequences of SEQ ID NO:    29, of SEQ ID NO: 30, of SEQ ID NO: 31, and of SEQ ID NO: 32.

Experimental Procedures

TABLE 1 Bispecific antibodies and their respective sequences DescriptionShort Name Sequences <VEGF-ANG-2> VEGFang2-0012 SEQ ID NO: 21, CrossMAbIgG1 with SEQ ID NO: 22, AAA mutations SEQ ID NO: 23, SEQ ID NO: 24<VEGF-ANG-2> VEGFang2-0201- SEQ ID NO: 39, CrossMAb IgG1 wild type SEQID NO: 40, (without AAA mutations) SEQ ID NO: 41, SEQ ID NO: 42<VEGF-ANG-2> VEGFang2-0016 SEQ ID NO: 25, CrossMAb IgG1 with SEQ ID NO:26, AAA mutations and SEQ ID NO: 27, P329G LALA mutations SEQ ID NO: 28<VEGF-ANG-2> VEGFang2-0015 SEQ ID NO: 43, CrossMAb IgG1 with SEQ ID NO:44, P329G LALA mutations SEQ ID NO: 45, only (without AAA SEQ ID NO: 46mutations) <VEGF-ANG-2> — SEQ ID NO: 29, CrossMAb IgG4 with SEQ ID NO:30, AAA mutations and with SEQ ID NO: 31, SPLE mutations SEQ ID NO: 32<VEGF-ANG-2> — SEQ ID NO: 33, OAscFab IgG1 with AAA SEQ ID NO: 34,mutations SEQ ID NO: 35 <VEGF-ANG-2> — SEQ ID NO: 36, OAscFab IgG4 withAAA SEQ ID NO: 37, mutations and with SPLE SEQ ID NO: 38 mutations

Please note that the term “with (the) mutations AAA” as used hereinrefers the mutations I253A (Ile253Ala), H310A (His310Ala), and H435A(His435Ala) in the constant heavy chain region of IgG1 or IgG4(numbering according to EU Index of Kabat), the term “with (the)mutations P329G LALA” as used herein refers to the mutations L234A(Leu235Ala) , L235A (Leu234Ala) and P329G (Pro329Gly) in the constantheavy chain region of IgG1 subclass (numbering according to EU Index ofKabat), and the term “with (the) mutations SPLE” as used herein refersto the S228P (Ser228Pro) and L235E (Leu235Glu) the constant heavy chainregion of IgG4 subclass (numbering according to EU Index of Kabat).

EXAMPLES

Materials & General Methods

General information regarding the nucleotide sequences of humanimmunoglobulin light and heavy chains is given in: Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Aminoacids of antibody chains are numbered and referred to according to EUnumbering (Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63 (1969)78-85; Kabat, E. A., et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991)).

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular Cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were ordered according to given specifications atGeneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences were determined by double strand sequencing performed atMediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH(Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wisconsin) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was usedfor sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

For the expression of the described antibodies, variants of expressionplasmids for transient expression (e.g. in HEK293-F) cells based eitheron a cDNA organization with or without a CMV-Intron A promoter or on agenomic organization with a CMV promoter were applied.

Beside the antibody expression cassette the vectors contained:

-   -   an origin of replication which allows replication of this        plasmid in E. coli,    -   a β-lactamase gene which confers ampicillin resistance in E.        coli., and    -   the dihydrofolate reductase gene from Mus musculus as a        selectable marker in eukaryotic cells    -   The transcription unit of the antibody gene was composed of the        following elements:    -   unique restriction site(s) at the 5′ end    -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   followed by the Intron A sequence in the case of the cDNA        organization,    -   a 5′-untranslated region of a human antibody gene,    -   an immunoglobulin heavy chain signal sequence,    -   the human antibody chain (wildtype or with domain exchange)        either as cDNA or as genomic organization with the        immunoglobulin exon-intron organization    -   a 3′ untranslated region with a polyadenylation signal sequence,        and    -   unique restriction site(s) at the 3′ end.

The fusion genes comprising the antibody chains as described below weregenerated by PCR and/or gene synthesis and assembled by knownrecombinant methods and techniques by connection of the accordingnucleic acid segments e.g. using unique restriction sites in therespective vectors. The subcloned nucleic acid sequences were verifiedby DNA sequencing. For transient transfections larger quantities of theplasmids were prepared by plasmid preparation from transformed E. colicultures (Nucleobond AX, Macherey-Nagel).

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

The bispecific antibodies were expressed by transient co-transfection ofthe respective expression plasmids in in HEK29-F cells growing insuspension as described below.

Example 1

Expression and Purification

Transient Transfections in HEK293-F System

The bispecific antibodies were generated by transient transfection withthe respective plasmids (e.g. encoding the heavy and modified heavychain, as well as the corresponding light and modified light chain)using the HEK293-F system (Invitrogen) according to the manufacturer'sinstruction. Briefly, HEK293-F cells (Invitrogen) growing in suspensioneither in a shake flask or in a stirred fermenter in serum-freeFreeStyle™ 293 expression medium (Invitrogen) were transfected with amix of the four expression plasmids and 293fectin™ or fectin(Invitrogen).

For 2 L shake flask (Corning) HEK293-F cells were seeded at a density of1.0E*6 cells/mL in 600 mL and incubated at 120 rpm, 8% CO2. The dayafter the cells were transfected at a cell density of ca. 1.5E*6cells/mL with ca. 42 mL mix of A) 20 mL Opti-MEM™ (Invitrogen) with 600μg total plasmid DNA (1 μg/mL) encoding the heavy or modified heavychain, respectively and the corresponding light chain in an equimolarratio and B) 20 ml Opti-MEM™+1.2 mL 293 fectin or fectin (2 μl/mL).According to the glucose consumption glucose solution was added duringthe course of the fermentation. The supernatant containing the secretedantibody was harvested after 5-10 days and antibodies were eitherdirectly purified from the supernatant or the supernatant was frozen andstored.

Purification

Bispecific antibodies were purified from cell culture supernatants byaffinity chromatography using MabSelectSure-Sepharose® (for non_AAAmutants) (GE Healthcare, Sweden) or kappaSelect-Agarose (for AAAmutants) (GE Healthcare, Sweden), hydrophobic interaction chromatographyusing butyl-Sepharose® (GE Healthcare, Sweden) and Superdex 200 sizeexclusion (GE Healthcare, Sweden) chromatography.

Briefly, sterile filtered cell culture supernatants were captured on aMabSelect SuRe™ resin equilibrated with PBS buffer (10 mM Na₂HPO₄, 1 mMKH₂PO₄, 137 mM NaCl and 2.7 mM KC1, pH 7.4), washed with equilibrationbuffer and eluted with 25 mM sodium citrate at pH 3.0. The AAA mutantswere captured on a kappaSelect resin equilibrated with 25 mM Tris, 50 mMNaCl, pH 7.2, washed with equilibration buffer and eluted with 25 mMsodium citrate pH 2.9. The eluted protein fractions were pooled andneutralized with 2M Tris, pH 9.0. The antibody pools were prepared forhydrophobic interaction chromatography by adding 1.6 M ammonium sulfatesolution to a final concentration of 0.8 M ammonium sulfate and the pHadjusted to pH 5.0 using acetic acid. After equilibration of thebutyl-Sepharose® resin with 35 mM sodium acetate, 0.8 M ammoniumsulfate, pH 5.0, the antibodies were applied to the resin, washed withequilibration buffer and eluted with a linear gradient to 35 mM sodiumacetate pH 5.0. The bispecific antibody containing fractions were pooledand further purified by size exclusion chromatography using a Superdex200 26/60 GL (GE Healthcare, Sweden) column equilibrated with 20 mMhistidine, 140 mM NaCl, pH 6.0. The bispecific antibody containingfractions were pooled, concentrated to the required concentration usingVivaspin ultrafiltration devices (Sartorius Stedim Biotech S.A., France)and stored at −80° C.

TABLE 2 Yields of bispecific <YEGF-ANG-2> antibodies VEGFang2-0015VEGFang2-0016 (with (without AAA mutation) AAA mutation) Titersupernatant   64 μg/ml, n.a. (2 L = 128 mg) (2 L scale) Protein A 118mg  n.a. (MabSelectSure) (~70% monomer) Kappa Select n.a. 117 mg (~83%monomer) Butyl Sepharose 60 mg  57 mg SEC 35 mg  38 mg (>95% monomer)(>95% monomer)

Purity and antibody integrity were analyzed after each purification stepby CE-SDS using microfluidic LabChip® technology (Caliper Life Science,USA). 5 μl of protein solution was prepared for CE-SDS analysis usingthe HT Protein Express

Reagent Kit according manufacturer's instructions and analysed onLabChip® GXII system using a HT Protein Express Chip. Data were analyzedusing LabChip® GX Software.

TABLE 3 Removal of typical side products by different sequentialpurification steps determined by CE-SDS. Purification Step VEGFang2-0015VEGFang2-0016 % peak area* * analysis: CE-SDS (Caliper Labchip GXII) ¾ ½¾ ½ (LC) mab ab (HC)2 ab (LC)2 LC mab ab (HC)2 ab 2 LC Mab 55.7 19 10.69.8 3.5 0.9 — Select Sure Kappa — 63 13.4 3.5 6.1 5.8 7.4 Select Butyl-81.4 1.9 2.3 8.2 3.6 1.8 76.2 1.3 0.7 8.3 7.7 5.8 Sepha- rose Super-92.4 1.8 2.6 1.4 0.5 0.5 99 1.1 n.d. n.d. n.d. n.d. dex 200_ SEC

The aggregate content of antibody samples was analyzed byhigh-performance SEC using a Superdex 200 analytical size-exclusioncolumn (GE Healthcare, Sweden) in 2xPBS (20 mM Na₂HPO₄, 2 mM KH₂PO₄, 274mM NaCl and 5.4 mM KCl, pH 7.4) running buffer at 25° C. 25 μg proteinwere injected on the column at a flow rate of 0.75 ml/min and elutedisocratic over 50 minutes.

Analogously the <VEGF-ANG-2> bispecific antibodies VEGFang2-0012 andVEGFang2-0201 were prepared and purified with the following yields:

VEGFang2-0012 VEGFang2-0201 (with AAA mutation) (without AAA mutation)Titer//amount —   36 μg/ml// 72 mg Scale 2.1 L   2 L Protein A — 66 mg(MabSelectSure) (~95% monomer) kappaSelect 43 mg — (~65% monomer) ButylSepharose — 45 mg SEC  14 mg 21 mg (>98% monomer) Yield 8.5 mghydoxylapatite (>98% monomer) Totatl yield 8.5 mg 21 mg (recovery) (20%)(30%)

Also the <VEGF-ANG-2> bispecific antibodies <VEGF-ANG-2> CrossMAb IgG4with AAA mutations and with SPLE mutations(SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32), <VEGF-ANG-2> OAscFab IgG1 with AAAmutations(SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35)and <VEGF-ANG-2>OAscFab IgG4 with AAA mutations and with SPLE mutations(SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 38) can be prepared and purified analogously.

Example 2

Analytics & Developability

Small-Scale DLS-Based Viscosity Measurement.

Viscosity measurement was essentially performed as described in (He, F.et al.,

Analytical Biochemistry 399 (2009) 141-3). Briefly, samples areconcentrated to various protein concentrations in 200 mM argininesuccinate, pH 5.5, before polystyrene latex beads (300 nm diameter) andPolysorbate 20 (0.02% v/v) are added. Samples are transferred into anoptical 384-well plate by centrifugation through a 0.4 μm filter plateand covered with paraffine oil. The apparent diameter of the latex beadsis determined by dynamic light scattering at 25° C. The viscosity of thesolution can be calculated as η=η0(rh/rh,0) (η: viscosity; η0: viscosityof water; rh: apparent hydrodynamic radius of the latex beads; rh,0:hydrodynamic radius of the latex beads in water.

To allow comparison of various samples at the same concentration,viscosity-concentration data were fitted with the Mooney equation(Equation 1) (Mooney, Colloid Sci, 1951; Monkos, Biochem. Biophys. Acta1997) and data interpolated accordingly.

$\begin{matrix}{\eta = {\eta_{0}{\exp \left( \frac{S\; \Phi}{1 - {K\Phi}} \right)}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

(S: hydrodynamic interaction parameter of the protein; K: self-crowdingfactor; Φ: volume fraction of the dissolved protein)

Results are shown in FIG. 2: VEGFang2-0016 with AAA mutations in the Fcpart shows a lower viscosity at all measured temperatures compared toVEGFang2-0015 without the AAA mutations in the Fc part.

DLS Aggregation Onset Temperature

Samples are prepared at a concentration of 1 mg/mL in 20 mMHistidine/Histidine chloride, 140 mM NaCl, pH 6.0, transferred into anoptical 384-well plate by centrifugation through a 0.4 μm filter plateand covered with paraffine oil. The hydrodynamic radius is measuredrepeatedly by dynamic light scattering while the samples are heated witha rate of 0.05° C./min from 25° C. to 80° C. The aggregation onsettemperature is defined as the temperature at which the hydrodynamicradius starts to increase. Results are shown in FIG. 3. In FIG. 3 theaggregation of VEGFang2-0015 without the AAA mutations versusVEGFang2-0016 with AAA mutations in the Fc part is shown. VEGFang2-0016showed a aggregation onset temperature of 61° C. whereas VEGFang2-0015without the AAA mutations showed a onset temperature of 60° C.

DLS Timecourse

Samples are prepared at a concentration of 1 mg/mL in 20 mMHistidine/Histidine chloride, 140 mM NaCl, pH 6.0, transferred into anoptical 384-well plate by centrifugation through a 0.4 μm filter plateand covered with paraffine oil. The hydrodynamic radius is measuredrepeatedly by dynamic light scattering while the samples are kept at aconstant temperature of 50° C. for up to 145 hours. In this experiment,aggregation tendencies of the native, unfolded protein at elevatedtemperature would lead to an increase of the average particle diameterover time. This DLS-based method is very sensitive for aggregatesbecause these contribute over-proportionally to the scattered lightintensity. Even after 145 hours at 50° C. (a temperature close to theaggregation-onset temperature, see above), an average particle diameterincrease of only less than 0.5 nm was found for both VEGFang2-0015 andVEGFang2-0016

7 day Storage at 40° C. at 100 mg/ml (HMW Increase)

Samples are concentrated to a final concentration of 100 mg/mL in 200 mMarginine succinate, pH 5.5, sterile filtered and quiescently stored at40° C. for 7 days. Before and after storage, the content of high and lowmolecular weight species (HMWs and LMWs, respectively) is determined bysize-exclusion chromatography. The difference in HMW and LMW contentbetween the stored sample and a sample measured immediately afterpreparation is reported as “HMW increase” and “LMW increase”,respectively. Results are shown in Table 4 and FIG. 4, which show thatVEGFang2-0015 (without AAA mutation) shows a higher reduction of themain peak and a higher HMW increase compared to VEGF Ang2-0016 (with AAAmutation). Surprisingly VEGF Ang2-0016 (with AAA mutation) showed alower aggregation tendency compared toVEGFang2-0015 (without AAAmutation).

TABLE 4 Delta Main-, HMW and LMW peaks after 7 d at 40° C. delta_area %(40° C.-(−80° C.)) Main Peak HMW LMW VEGFang2-0015 −3.56 2.89 0.67 (−AAAmutations) VEGFang2-0016 −1.74 1.49 0.25 (+AAA mutations)

The functional analysis of anti-VEGF and anti-Ang2 bispecific antibodieswas assessed by Surface Plasmon Resonance (SPR) using a BlAcore® T100 orT200 instrument (GE Healthcare) at 25° C. The BIAcore® system is wellestablished for the study of molecule interactions. SPR-technology isbased on the measurement of the refractive index close to the surface ofa gold coated biosensor chip. Changes in the refractive index indicatemass changes on the surface caused by the interaction of immobilizedligand with analyte injected in solution. The mass increases ifmolecules bind immobilized ligands on the surface, and vice versa, themass decreases in case of dissociation of the analyte from theimmobilized ligand (reflecting complex dissociation). SPR allows acontinuous real-time monitoring of ligand/analyte binding and thus thedetermination of the association rate constant (ka), the dissociationrate constant (kd), and of the equilibrium constant (KD).

Example 3

Binding to VEGF, Ang2, FcgammaR and FcRn

VEGF Isoforms Kinetic Affinity Including Assessment ofSpecies-Crossreactivity

Around 12000 resonance units (RU) of the capturing system (10 μg/ml goatanti human F(ab)′₂; Order Code: 28958325; GE Healthcare Bio-Sciences AB,Sweden) were coupled on a CMS chip (GE Healthcare BR-1005-30) at pH 5.0by using an amine coupling kit supplied by the GE Healthcare. The sampleand system buffer was PBS-T (10 mM phosphate buffered saline including0.05% Tween20) pH 7.4. The flow cell was set to 25° C.—and the sampleblock set to 12° C.—and primed with running buffer twice. The bispecificantibody was captured by injecting a 50 nM solution for 30 sec at a flowof 5 μl/min. Association was measured by injection of human hVEGF121,mouse mVEGF120 or rat rVEGF164 in various concentrations in solution for300 sec at a flow of 30 μl/min starting with 300 nM in 1:3 dilutions.The dissociation phase was monitored for up to 1200 sec and triggered byswitching from the sample solution to running buffer. The surface wasregenerated by 60 sec washing with a Glycine pH 2.1 solution at a flowrate of 30 μl/min. Bulk refractive index differences were corrected bysubtracting the response obtained from a goat anti human F(ab′)₂surface. Blank injections are also subtracted (=double referencing). Forcalculation of apparent K_(D) and other kinetic parameters the Langmuir1:1 model was used. Results are shown in Table 5.

Ang2 Solution Affinity Including Assessment of Species-Crossreactivity

Solution affinity measures the affinity of an interaction by determiningthe concentration of free interaction partners in an equilibriummixture. The solution affinity assay involves the mixing of an<VEGF-ANG-2> bispecific antibody, kept at a constant concentration, witha ligand (=Ang2) at varying concentrations. Maximum possible resonanceunits (e.g. 17000 resonance units (RU)) of an antibody was immobilizedon the CMS chip (GE Healthcare BR-1005-30) surface at pH 5.0 using anamine coupling kit supplied by the GE Healthcare. The sample and systembuffer was HBS-P pH 7.4. Flow cell was set to 25° C. and sample block to12° C. and primed with running buffer twice. To generate a calibrationcurve increasing concentrations of Ang2 were injected into a BlAcoreflowcell containing the immobilized VEGF-ANG-2> bispecific antibody. Theamount of bound Ang2 was determined as resonance units (RU) and plottedagainst the concentration. Solutions of each ligand (11 concentrationsfrom 0 to 200 nM for the VEGF-ANG-2> bispecific antibody) were incubatedwith 10 nM Ang2 and allowed to reach equilibrium at room temperature.Free Ang2 concentrations were determined from calibration curvegenerated before and after measuring the response of solutions withknown amounts of Ang2. A 4-parameter fit was set with XLfit4 (IDBSSoftware) using Model 201 using free Ang2 concentration as y-axis andused concentration of antibody for inhibition as x-axis. The affinitywas calculated by determining the inflection point of this curve. Thesurface was regenerated by one time 30 sec washing with a 0.85% H₃PO₄solution at a flow rate of 30 μl/min. Bulk refractive index differenceswere corrected by subtracting the response obtained from a blank-coupledsurface. Results are shown in Table 6.

FeRn Steady State Affinity

For FcRn measurement a steady state affinity was used to comparebispecific antibodies against each other. Human FcRn was diluted intocoupling buffer (10 μg/ml, Na-Acetate pH5.0) and immobilized on aC1-Chip (GE Healthcare BR-1005-35) by targeted immobilization procedureusing a BlAcore wizard to a final response of 200 RU. Flow cell was setto 25° C. and sample block to 12° C. and primed with running buffertwice. The sample and system buffer was PBS-T (10 mM phosphate bufferedsaline including 0.05% Tween20) pH 6.0. To assess different IgGconcentrations for each antibody, a concentration of 62.5 nM, 125 nM and250 nM, 500 nM was prepared. Flow rate was set to 30 μl/min and thedifferent samples were injected consecutively onto the chip surfacechoosing 180 sec association time. The surface was regenerated byinjected PBS-T pH 8 for 60 sec at a flow rate of 30 μl/min. Bulkrefractive index differences were corrected by subtracting the responseobtained from a blank surface. Buffer injections are also subtracted(=double referencing). For calculation of steady state affinity themethod from the Bia-Evaluation software was used. Briefly, the RU values(RU max) were plotted against the analysed concentrations, yielding adose-response curve. Based on a 2-parametric fit, the upper asymptote iscalculated, allowing the determination of the half-maximal RU value andhence the affinity. Results are shown in FIG. 5 and Table 7. Analogouslythe affinity to cyno, mouse and rabbit FcRn can be determined.

FcgammaRIIIa Measurement

For FcgammaRIIIa measurement a direct binding assay was used. Around3000 resonance units (RU) of the capturing system (1 μg/ml Penta-His;Quiagen) were coupled on a CMS chip (GE Healthcare BR-1005-30) at pH 5.0by using an amine coupling kit supplied by the GE Healthcare. The sampleand system buffer was HBS-P+pH 7.4. The flow cell was set to 25° C.—andsample block to 12° C.—and primed with running buffer twice. TheFcgammaRIIIa -His-receptor was captured by injecting a 100 nM solutionfor 60 sec at a flow of 5 μl/min. Binding was measured by injection of100 nM of bispecific antibody or monospecific control antibodies(anti-Dig for IgG1 subclass and an IgG4 subclass antibody) for 180 secat a flow of 30 μl/. The surface was regenerated by 120 sec washing withGlycine pH 2.5 solution at a flow rate of 30 μl/min. BecauseFcgammaRIIIa binding differs from the Langmuir 1:1 model, onlybinding/no binding was determined with this assay. In a similar mannerFcgammaRIa , and FcgammaRIIa binding can be determined. Results areshown in FIG. 6, where it follows that by introduction of the mutationsP329G LALA no more binding to FcgammaRIIIa could be detected.

Assessment of Independent VEGF- and Ang2-Binding to the <VEGF-ANG-2>Bispecific Antibodies

Around 3500 resonance units (RU) of the capturing system (10 μg/ml goatanti human IgG; GE Healthcare Bio-Sciences AB, Sweden) were coupled on aCM4 chip (GE Healthcare BR-1005-34) at pH 5.0 by using an amine couplingkit supplied by the GE Healthcare. The sample and system buffer wasPBS-T (10 mM phosphate buffered saline including 0.05% Tween20) pH 7.4.The temperature of the flow cell was set to 25° C. and of the sampleblock to 12° C. Before capturing, the flow cell was primed with runningbuffer twice.

The bispecific antibody was captured by injecting a 10 nM solution for60 sec at a flow of 5 μl/min. Independent binding of each ligand to thebispecific antibody was analysed by determining the active bindingcapacity for each ligand, either added sequentially or simultaneously(flow of 30 μl/min):

-   -   1. Injection of human VEGF with a concentration of 200 nM for        180 sec (identifies the single binding of the antigen).    -   2. Injection of human Ang2 with a concentration of 100 nM for        180 sec (identifies single binding of the antigen).    -   3. Injection of human VEGF with a concentration of 200 nM for        180 sec followed by an additional injection of human Ang2 with a        concentration of 100 nM for 180 sec (identifies binding of Ang2        in the presence of VEGF).    -   4. Injection of human Ang2 with a concentration of 100 nM for        180 sec followed by an additional injection of human VEGF with a        concentration of 200 nM (identifies binding of VEGF in the        presence of Ang2).    -   5. Co-Injection of human VEGF with a concentration of 200 nM and        of human Ang2 with a concentration of 100 nM for 180 sec        (identifies the binding of VEGF and of Ang2 at the same time).

The surface was regenerated by 60 sec washing with a 3 m MgCl2 solutionat a flow rate of 30 μl/min. Bulk refractive index differences werecorrected by subtracting the response obtained from a goat anti humanIgG surface.

The bispecific antibody is able to bind both antigens mutualindependently if the resulting final signal of the approaches 3, 4 & 5equals or is similar to the sum of the individual final signals of theapproaches 1 and 2. Results are shown in Table 9, where both antibodiesVEGFang2-0016, VEGFang2-0012 are shown to be able to bind mutualindependently to VEGF and ANG2

Assessment of Simultaneous VEGF- and Ang2-binding to the <VEGF-ANG-2>Bispecific Antibodies

First, around 1600 resonance units (RU) of VEGF (20 μg/ml) were coupledon a CM4 chip (GE Healthcare BR-1005-34) at pH 5.0 by using an aminecoupling kit supplied by the GE Healthcare. The sample and system bufferwas PBS-T (10 mM phosphate buffered saline including 0.05% Tween 20) pH7.4. Flow cell was set to 25° C. and sample block to 12° C. and primedwith running buffer twice. Second, 50 nM solution of the bispecificantibody was injected for 180 sec at a flow of 30 μl/min. Third, hAng-2was injected for 180 sec at a flow of 30 μl/min. The binding response ofhAng-2 depends from the amount of the bispecific antibody bound to VEGFand shows simultaneous binding. The surface was regenerated by 60 secwashing with a 0.85% H3PO4 solution at a flow rate of 30 μl/min.Simultaneous binding is shown by an additional specific binding signalof hAng2 to the previous VEGF bound <VEGF-ANG-2> bispecific antibodies.For both bispecific antibodies VEGFang2-0015 and VEGFang2-0016simultaneous VEGF- and Ang2-binding to the <VEGF-ANG-2> bispecificantibodies could be detected (data not shown).

TABLE 5 Results: Kinetic affinities to VEGF isoforms from differentspecies VEGFang2- VEGFang2- VEGFang2- VEGFang2- 0015- 0016- 0012- 0201-apparent apparent apparent apparent affinity affinity affinity affinityHuman VEGF ≤1 pM ≤1 pM ≤1 pM ≤1 pM 121 (out of (out of (out of (out ofBiacore Biacore Biacore Biacore specification) specification)specification) specification) mouseVEGF no binding no binding no bindingno binding 120 Rat VEGF 13 nM 14 nM 24 nM 35 nM 164

TABLE 6 Results: Solution affinities to Ang2 VEGFang2- VEGFang2-VEGFang2- VEGFang2-- 0015 KD 0016 KD 0012 KD 0201 KD [nM] [nM] [nM] [nM]humanAng2 8 20 20 tbd cynoAng2 5 13 10 tbd mouseAng2 8 13 8 tbdrabbitAng2 4 11 8 tbd

TABLE 7 Results: Affinity to FcRn of <VEGF-ANG-2> bispecific antibodiesVEGFang2- VEGFang2- VEGFang2- VEGFang2-- 0015 0016 0012 0201 [affinity][affinity] [affinity] [affinity] Human 0.8 μM no binding no binding 0.8μM FcRn Cyno 0.9 μM no binding no binding 1.0 μM FcRn Mouse 0.2 μM nobinding no binding 0.2 μM FcRn

TABLE 8 Results Binding to FcgammaRI-IIIa VEGFang2- VEGFang2- VEGFang2-VEGFang2- 0015 0016 0012 0201 FcyRIa No binding No binding BindingBinding FcyRIIa No binding No binding No binding Binding FcyRIIIa Nobinding No binding No binding Binding

TABLE 9 Results: Independent binding of VEGF-and Ang2 to <VEGF-ANG-2>bispecific antibodies 3) first 4) first 5) VEGF Ang2 Coinjection thenthen Ang2 + 1) Ang2 2) VEGF Ang2 VEGF VEGF [RUmax] [RUmax] [RUmax][RUmax] [RUmax] VEGFang2- 174 50 211 211 211 0016 VEGFang2- 143 43 178177 178 0012

Example 4

Mass Spectrometry

This section describes the characterization of <VEGF-ANG-2> bispecificantibodies with emphasis on the correct assembly. The expected primarystructures were confirmed by electrospray ionization mass spectrometry(ESI-MS) of the deglycosylated, and intact or IdeS-digested(IgG-degrading enzyme of S. pyogenes) <VEGF-ANG-2> bispecificantibodies. The IdeS-digestion was performed with 100 μg purifiedantibody incubated with 2 μg IdeS protease (Roche) in 100 mmol/LNaH₂PO₄/Na₂HPO₄, pH 7.1 at 37° C. for 5 h. Subsequently, the antibodieswere deglycosylated with N-Glycosidase F, Neuraminidase andO-glycosidase (Roche) in 100 mmol/L NaH₂PO₄/Na₂HPO₄, pH 7.1 at 37° C.for up to 16 h at a protein concentration of 1 mg/ml and subsequentlydesalted via HPLC on a Sephadex G25 column (GE Healthcare). The totalmass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (BrukerDaltonik) equipped with a TriVersa NanoMate® source (Advion).

The masses obtained for the IdeS-digested, deglycosylated (Table 10), orintact, deglycosylated (Table 11) molecules correspond to the predictedmasses deduced from the amino acid sequences for the <VEGF-ANG-2>bispecific antibodies consisting of two different light chains LC_(Ang2)and LC_(Lucentis), and two different heavy chains HC_(Ang2) andHC_(Lucentis).

TABLE 10 Masses of the deglycosylated and IdeS-digested bispecific<VEGF/ANG2> antibodies VEGFang2-0201 (without AAA mutation)andVEGFang2-0012 (with AAA mutation) F(ab′)2 of the VEGF- DeglycosylatedFc of ANG-2> bispecific the VEGF-ANG-2> antibody bispecific antibodyPredicted Observed Predicted Observed Average Average Average AverageMass Mass Mass Mass Sample [Da] [Da] [Da] [Da] VEGFang2- 99360.8 99360.747439.2 47430.1 0201 VEGFang2- 99360.8 99361.1 47087.7 47082.0 0012

TABLE 11 Masses of the deglycosylated <VEGF/ANG2> antibodiesVEGFang2-0016 (with AAA mutation) and VEGFang2-0015 (without AAAmutation) Deglycosylated VEGF-ANG-2> bispecific antibody PredictedObserved Average Average Mass Mass [Da] [Da] VEGFang2- 146156.9 146161.20016 VEGFang2- 146505.3 146509.4 0015

Example 5

Fc-Rn Chromatography

Coupling to Streptavidin Sepharose:

One gram streptavidin Sepharose® (GE Healthcare) was added to thebiotinylated and dialyzed receptor and incubated for two hours withshaking. The receptor derivatized sepharose was filled in a 1 ml XKcolumn (GE Healthcare).

Chromatography Using the FcRn Affinity Column:

Conditions:

column dimensions: 50 mm×5 mm

bed height: 5 cm

loading: 50 μg sample

equilibration buffer: 20 mM MES, with 150 mM NaCl, adjusted to pH 5.5

elution buffer: 20 mM Tris/HC1, with 150 mM NaCl, adjusted to pH 8.8

elution: 7.5 CV equilibration buffer, in 30 CV to 100% elution buffer,10 CV elution buffer

Hu FcRn Affinity Column Chromatography

In the following table retention times of <VEGF-ANG-2> bispecificantibodies on affinity columns comprising human FcRn are given. Datawere obtained using the conditions above. In the following Tableretention times of <VEGF-ANG-2> bispecific antibodies on human FcRn aregiven.

TABLE 12 Results: retention times of <VEGF-ANG-2> bispecific antibodiesretention time antibody [min] VEGFAng2-0015 78.5 (without AAA mutation)VEGFAng2-0201 78.9 (without AAA mutation) VEGFAng2-0012 2.7 (Void-peak)(with AAA mutation) VEGFAng2-0016 2.7 (Void-peak) (with AAA mutation)

Example 6

Pharmacokinetic(PK) Properties

PK Data with Fc-Rn Mice Transgenic for Human FcRn

In Life Phase

The study included female C57BL/6J mice (background); mouse FcRndeficient, but hemizygous transgenic for human FcRn (huFcRn, line276-/tg)

Part 1

All mice were injected once intravitreally into the right eye with 2μL/animal of the appropriate solution (i.e. 21 μg compound/animal(VEGFAng2-0015 (without AAA mutation) or 23.6 μg compound/animal(VEGFAng2-0016 (with AAA mutation).

Mice were allocated to 2 groups with 6 animals each. Blood samples aretaken from group 1 at 2, 24 and 96 hours and from group 2 at 7, 48 and168 hours after dosing.

Injection into the vitreous of the right mouse eye was performed byusing the NanoFil Microsyringe system for nanoliter injection from WorldPrecision

Instruments, Inc., Berlin, Germany. Mice were anesthetized with 2.5%Isoflurane and for visualization of the mouse eye a Leica MZFL 3microscope with a 40 fold magnification and a ring-light with a Leica KL2500 LCD lightning was used. Subsequently, 2 μL of the compound wereinjected using a 35-gauge needle.

Blood was collected via the retrobulbar venous plexus of thecontralateral eye from each animal for the determination of the compoundlevels in serum.

Serum samples of at least 50 μl were obtained from blood after 1 hour atRT by centrifugation (9300 xg) at 4° C. for 3 min. Serum samples werefrozen directly after centrifugation and stored frozen at −80° C. untilanalysis. Treated eyes of the animals of group 1 were isolated 96 hoursafter treatment and of the animals of group 2 168 hours after treatment.Samples were stored frozen at −80° C. until analysis.

Part 2

All mice were injected once intravenously via the tail vein with 200μL/animal of the appropriate solution (i.e. 21 μg compound/animal(VEGFAng2-0015 (without AAA mutation) or 23.6 μg compound/animal(VEGFAng2-0016 (with AAA mutation).

Mice were allocated to 2 groups with 5 animals each. Blood samples aretaken from group 1 at 1, 24 and 96 hours and from group 2 at 7, 48 and168 hours after dosing. Blood was collected via the retrobulbar venousplexus from each animal for the determination of the compound levels inserum.

Serum samples of at least 50 μl were obtained from blood after 1 hour atRT by centrifugation (9300 xg) at 4° C. for 3 min. Serum samples werefrozen directly after centrifugation and stored frozen at −80° C. untilanalysis.

Preparation of Whole Eye Lysates (Mice)

The eye lysates were gained by physico-chemical disintegration of thewhole eye from laboratory animals. For mechanical disruption, each eyewas transferred into a 1.5-mL micro vial with conical bottom. Afterfreeze and thawing, the eyes were washed with 1 mL cell washing bufferonce (Bio-Rad, Bio-Plex Cell Lysis Kit, Cat. No. 171-304011). In thefollowing step, 5004 of freshly prepared cell lysis buffer were addedand the eyes were grinded using a 1.5 mL tissue grinding pestle (KimbleChase, 1.5 mL pestle, Art. No. 749521-1500). The mixture was then frozenand thawed five times and grinded again. To separate lysate fromremaining tissue the samples were centrifuged for 4 min at 4500 g. Aftercentrifuging the supernatant was collected and stored at −20° C. untilfurther analysis in the quantification ELISA.

Analysis

The concentrations of the <VEGF/ANG2> antibodies in mice serum and eyelysates were determined with an enzyme linked immunosorbent assay(ELISA)

For quantification of <VEGF/ANG2> antibodies in mouse serum samples andeye lysates, a standard solid-phase serial sandwich immunoassay withbiotinylated and digoxigenated monoclonal antibodies used as capture anddetection antibodies was performed. To verify the integrity of thebispecifity of the analyte the biotinylated capture antibody recognizesthe anti-VEGF-binding site whereas the digoxigenated detection antibodywill bind to the anti-Ang2 binding site of the analyte. The bound immunecomplex of capture antibody, analyte and detection antibody on the solidphase of the streptavidin coated micro titer plate (SA-MTP) is thendetected with a horseradish-peroxidase coupled to an anti-digoxigeninantibody. After washing unbound material from the SA-MTP and addition ofABTS-substrate, the gained signal is proportional to the amount ofanalyte bound on the solid phase of the SA-MTP. Quantification is thendone by converting the measured signals of the samples intoconcentrations referring to calibrators analyzed in parallel.

In a first step the SA-MTP was coated with 100 μL/well of biotinylatedcapture antibody solution (mAb<Id<VEGF>>M-2.45.51-IgG-Bi(DDS)) with aconcentration of 1 μg/mL for one hour at 500 rpm on a MTP-shaker.Meanwhile calibrators, QC-samples and samples were prepared. Calibratorsand QC-samples are diluted to 2% serum matrix; samples were diluteduntil the signals were within the linear range of the calibrators.

After coating the SA-MTP with capture antibody, the plate was washedthree times with washing buffer and 300 μL/well. Subsequently 100μl/well of the calibrators, QC-samples and samples were pipetted on theSA-MTP and incubated again for one hour at 500 rpm. The analyte was nowbound with its anti-VEGF binding site via the capture antibody to thesolid phase of the SA-MTP. After incubation and removal of unboundanalyte by washing the plate 100 μL/well of the first detection antibody(mAb<Id-<Ang2>>M-2.6.81-IgG-Dig(XOSu)) with a concentration of 250 ng/mLwas added to the SA-MTP. Again, the plate was incubated for one hour at500 rpm on a shaker. After washing, 100 μL/well of the second detectionantibody (pAb<Digoxigenin>S-Fab-POD (poly)) at a concentration of 50mU/mL was added to the wells of the SA-MTP and the plate was incubatedagain for one hour at 500 rpm. After a final washing step to removeexcess of detection antibody, 100 μL/well substrate (ABTS) is added. Theantibody-enzyme conjugate catalyzes the color reaction of the ABTS®substrate. The signal was then measured by an ELISA reader at 405 nmwavelength (reference wavelength: 490 nm ([405/490] nm)).

Pharmacokinetic Evaluation

The pharmacokinetic parameters were calculated by non-compartmentalanalysis, using the pharmacokinetic evaluation program WinNonlin™(Pharsight), version 5.2.1.

Results: A) Serum Concentrations

Results for serum concentrations are shown in Tables 13 to 16 and FIG.7B to 7C

TABLE 13 VEGFAng2-0015 (without AAA mutation): Comparison of serumconcentrations after intravitreal and intravenous application Serumconcentration Serum concentration after intravitreal after intravenousapplication application Average conc. Average conc. ID [μg/mL] [μg/mL] 1 h 17.7  2 h 9.8  7 h 10.4 12.1  24 h 6.4 8.3  48 h 6.5 6.9  96 h 3.44.1 168 h 2.9 2.7

TABLE 14 VEGFAng2-0016 (with AAA mutation): Comparison of serumconcentrations after intravitreal and intravenous application Serumconcentration Serum concentration after intravitreal after intravenousapplication application Average conc. Average conc. ID [μg/mL] [μg/mL] 1 h 18.4  2 h 7.0  7 h 8.7 10.0  24 h 2.2 3.3  48 h 1.0 1.0  96 h 0.10.1 168 h 0.0 0.0

TABLE 15 VEGFang2-0015 (without AAA mutation) and VEGFang2-0016 (withAAA mutation): Comparison of serum concentrations after intravitrealapplication) VEGFang2-0015 VEGFang2-0016 (without AAA mutation) (withAAA mutation) Average conc. Average conc. ID [μg/mL] [μg/mL]  2 h 9.87.0  7 h 10.4 8.7  24 h 6.4 2.2  48 h 6.5 1.0  96 h 3.4 0.1 168 h 2.90.0

TABLE 16 VEGFang2-0015 (without AAA mutation) and VEGFang2-0016 (withAAA mutation): Comparison of serum concentrations after intravenousapplication VEGFang2-0015 VEGFang2-0016 (without AAA mutation) (with AAAmutation) Average conc. Average conc. ID [μg/mL] [μg/mL]  1 h 17.7 18.4 7 h 12.1 10.0  24 h 8.3 3.3  48 h 6.9 1.0  96 h 4.1 0.1 168 h 2.7 0.0

Results: B) Concentrations in Eye-Lysates of Left and Right Eyes

Results for concentrations in eye lysates are shown in Tables 17 to 18and FIGS. 7D to 7E

TABLE 17 a: Concentrations of VEGFang2-0015 (without AAA mutation) ineye lysates after intra vitreal application into right eye Mean conc.values from n = 6 mice ID mean conc. [ng/mL]  96 h Left eye 8.7 Righteye 46.1 168 h Left eye 4.3 Right eye t 12.9 b: Concentrations ofVEGFang2-0015 (without AAA mutation) in eye lysates after intravenousapplication Mean conc. values from n = 5 mice ID mean conc. [ng/mL]  96h Left eye 4.2 Right eye 7.5 168 h Left eye 3.4 Right eye 6.1

TABLE 18 a: Concentrations of VEGFang2-0016 (with AAA mutation) in eyelysates after intra vitreal application into right eye Mean conc. valuesfrom n = 5 mice ID mean conc. [ng/mL]  96 h Left eye 0.3 Right eye 34.5168 h Left eye 0.1 Right eye 9.0 b: Concentrations of VEGFang2-0016(with AAA mutation) in eye lysates after intravenous application Meanconc. values from n = 5 mice ID mean conc. [ng/mL]  96 h Left eye 0.0Right eye 0.1 168 h Left eye 0.0 Right eye 0.1

Summary of Results:

After intravitreal application the bispecific <VEGF/ANG2> antibodyaccording to the invention VEGFang2-0016 (with AAA mutation) showssimilar concentrations (after 96 and 168 hours) in the eye lysates ascompared to the bispecific <VEGF/ANG2> antibody without AAA mutationVEGFang2-0015.

Also after intravitreal application the bispecific <VEGF/ANG2> antibodyaccording to the invention VEGFang2-0016 (with AAA mutation) shows inaddition a faster clearance and shorter half-life in the serum ascompared to the bispecific <VEGF/ANG2> antibody without AAA mutationVEGFang2-0015.

Example 7

Mouse Cornea Micropocket Angiogenesis Assay

To test the anti-angiogenic effect bispecific <VEGF/ANG2> antibody withthe respective anti-VEGF VH and VL of SEQ ID NO: 7 and 8 and theanti-ANG2 VH and VL of SEQ ID NO: 15 and 16 on VEGF-induced angiogenesisin vivo, we perform the mouse corneal angiogenesis assay. In this assaya VEGF soaked Nylaflo® disc is implanted into a pocket of the avascularcornea at a fixed distance to the limbal vessels. Vessels immediatelygrow into the cornea towards the developing VEGF gradient. 8 to 10 weeksold female Balb/c mice were purchased from Charles River, Sulzfeld,Germany. The protocol is modified according to the method described byRogers, M. S., et al., Nat. Protoc. 2 (2007) 2545-2550. Briefly,micropockets with a width of about 500 μm are prepared under amicroscope at approximately 1 mm from the limbus to the top of thecornea using a surgical blade and sharp tweezers in the anesthetizedmouse. The disc (Nylaflo®, Pall Corporation, Michigan) with a diameterof 0.6 mm is implanted and the surface of the implantation area wassmoothened. Discs are incubated in corresponding growth factor or invehicle for at least 30 min. After 3, 5 and 7 days (or alternativelyonly after 3, 5 or 7 days) , eyes are photographed and vascular responseis measured. The assay is quantified by calculating the percentage ofthe area of new vessels per total area of the cornea.

The discs are loaded with 300 ng VEGF or with PBS as a control andimplanted for 7 days. The outgrowth of vessels from the limbus to thedisc is monitored over time on day 3, 5 and/or 7. One day prior to discimplantation the antibodies are administered intravenously at a dose of10 mg/kg (due to the intravenous application the serum-stableVEGFang2-0015 (without AAA mutation) which only differs fromVEGFang2-0016 by the AAA mutation and has the same anti-VEGF andanti-ANG2 VHs and VLs to mediate efficacy, is used as surrogate) fortesting the anti-angiogenic effect on VEGF-induced angiogenesis in vivo.Animals in the control group receive vehicle. The application volume is10 ml/kg.

1. A method for the reduction of the viscosity of an antibody whereinthe antibody comprises a constant heavy chain region of human IgG1 orhuman IgG4 subclass(derived from human origin and) wherein the methodcomprises the modification of the antibody constant heavy chain regionof human IgG1 or human IgG4 subclass with the mutations I253A, H310A,and H435A (numbering according to EU Index of Kabat).
 2. The method ofclaim 1, wherein the antibody is a bispecific antibody comprising afirst antigen-binding site that specifically binds to human VEGF and asecond antigen-binding site that specifically binds to human ANG-2,wherein i) said first antigen-binding site specifically binding to VEGFcomprises in the heavy chain variable domain a CDR3H region of SEQ IDNO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ IDNO:3, and in the light chain variable domain a CDR3L region of SEQ IDNO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6;and ii) said second antigen-binding site specifically binding to ANG-2comprises in the heavy chain variable domain a CDR3H region of SEQ IDNO: 9, a CDR2H region of, SEQ ID NO: 10, and a CDR1H region of SEQ IDNO: 11, and in the light chain variable domain a CDR3L region of SEQ IDNO: 12, a CDR2L region of SEQ ID NO: 13, and a CDR1L region of SEQ IDNO: 14, and wherein iii) the bispecific antibody comprises a constantheavy chain region of human IgG1 or human IgG4 subclass (derived fromhuman origin and) comprising the mutations I253A, H310A, and H435A(numbering according to EU Index of Kabat).
 3. The method of claim 2,wherein the bispecific antibody comprises a constant heavy chain regionof human IgG1 subclass (derived from human origin and) comprising themutations I253A, H310A, and H435A (numbering according to EU Index ofKabat) and further comprising the mutations L234A , L235A and P329G(numbering according to EU Index of Kabat).
 4. An antibody obtained bythe method of any one of claims 1 to
 3. 5. A bispecific antibodycomprising a first antigen-binding site that specifically binds to humanVEGF and a second antigen-binding site that specifically binds to humanANG-2, wherein i) said first antigen-binding site specifically bindingto VEGF comprises in the heavy chain variable domain a CDR3H region ofSEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQID NO:3, and in the light chain variable domain a CDR3L region of SEQ IDNO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6;and ii) said second antigen-binding site specifically binding to ANG-2comprises in the heavy chain variable domain a CDR3H region of SEQ IDNO: 9, a CDR2H region of, SEQ ID NO: 10, and a CDR1H region of SEQ IDNO: 11, and in the light chain variable domain a CDR3L region of SEQ IDNO: 12, a CDR2L region of SEQ ID NO: 13, and a CDR1L region of SEQ IDNO: 14, and wherein iii) the bispecific antibody comprises a constantheavy chain region of human IgG1 or human IgG4 subclass (derived fromhuman origin and) comprising the mutations I253A, H310A, and H435A(numbering according to EU Index of Kabat)
 6. The bispecific antibodyaccording to claim 5, wherein i) said first antigen-binding sitespecifically binding to VEGF comprises as heavy chain variable domain VHan amino acid sequence of SEQ ID NO: 7, and as light chain variabledomain VL an amino acid sequence of SEQ ID NO: 8, and ii) said secondantigen-binding site specifically binding to ANG-2 comprises as heavychain variable domain VH an amino acid sequence of SEQ ID NO: 15, and aslight chain variable domain VL an amino acid sequence of SEQ ID NO: 16.7. The bispecific antibody according to any one of claims 5 to 6,wherein the constant heavy chain region under iii) is of IgG1 subclass8. The bispecific antibody according to claim 6, wherein the constantheavy chain region of IgG1 subclass further comprises the mutationsL234A , L235A and P329G (numbering according to EU Index of Kabat) 9.The bispecific antibody according to any one of claims 5 to 6, whereinthe constant heavy chain region under iii) is of IgG4 subclass
 10. Thebispecific antibody according to claim 9, wherein the constant heavychain region of IgG4 subclass further comprises the mutations S228P andL235E (numbering according to EU Index of Kabat)
 11. The bispecificantibody according to claim 9, wherein the constant heavy chain regionof IgG4 subclass further comprises the mutations S228P , L235E and P329G(numbering according to EU Index of Kabat)
 12. A pharmaceuticalcomposition comprising an antibody according to any one of claims 4 and5 to
 11. 13. A nucleic acid encoding a bispecific antibody according toany one of claims 5 to
 11. 14. Expression vector containing said nucleicacid according claim 13 capable of expressing said nucleic acid in aprokaryotic or eukaryotic host cell.
 15. A prokaryotic or eukaryotichost cell comprising a vector according to claim
 14. 16. A method forthe preparation of a bispecific antibody according to any one of claims5 to 11 comprising the steps of a) transforming a host cell with vectorscomprising nucleic acid molecules encoding said antibody; b) culturingthe host cell under conditions that allow synthesis of said antibodymolecule; and c) recovering said antibody molecule from said culture.17. A bispecific antibody obtained by the method of claim
 16. 18. Abispecific, bivalent antibody comprising a first antigen-binding sitethat specifically binds to human VEGF and a second antigen-binding sitethat specifically binds to human ANG-2, characterized in comprising theamino acid sequences of SEQ ID NO: 25, of SEQ ID NO: 26, of SEQ ID NO:27, and of SEQ ID NO:
 28. 19. A bispecific, bivalent antibody comprisinga first antigen-binding site that specifically binds to human VEGF and asecond antigen-binding site that specifically binds to human ANG-2,characterized in comprising the amino acid sequences of SEQ ID NO: 21,of SEQ ID NO: 22., of SEQ ID NO: 23., and of SEQ ID NO:
 24. 20. Abispecific, bivalent antibody comprising a first antigen-binding sitethat specifically binds to human VEGF and a second antigen-binding sitethat specifically binds to human ANG-2, characterized in comprising theamino acid sequences of SEQ ID NO: 29, of SEQ ID NO: 30, of SEQ ID NO:31, and of SEQ ID NO:
 32. 21. A method of treatment of patient sufferingfrom ocular vascular diseases by administering an antibody according toany one of claims 4 and 5 to 11 to a patient in the need of suchtreatment.
 22. The method of claim 21, wherein the antibody isadministered via intravitreal application.